When he was a teenager, Paisley decided to follow his father and become a Christian minister. He delivered his first sermon aged 16 in a mission hall in County Tyrone. In the late 1940s he undertook theological training at the Barry School of Evangelism (now called the Wales Evangelical School of Theology), and later, for a year, at the Reformed Presbyterian Theological Hall in Belfast.

He became a Protestant evangelical minister in 1946 and remained one for the rest of his life. In 1951 he co-founded the fundamentalist Free Presbyterian Church of Ulster and was its leader until 2008. Paisley became known for his fiery sermons and regularly preached and protested against Roman Catholicism, ecumenism and homosexuality. He gained a large group of followers who were referred to as Paisleyites.

In 1949, Paisley formed a Northern Irish branch of the National Union of Protestants, the group being led in the UK by his uncle, W. St Clair Taylor. Paisley's first political involvement came at the 1950 general election, when he campaigned on behalf of the successful Ulster Unionist Party (UUP) candidate in Belfast West, the Church of Ireland minister James Godfrey MacManaway. Independent Unionist MP Norman Porter came to lead the National Union of Protestants, while Paisley became treasurer, but Paisley left after Porter refused to join the Free Presbyterian Church.

Paisley became involved in Ulster unionist/loyalist politics in the late 1950s. In the mid-late 1960s, he led and instigated loyalist opposition to the Catholic civil rights movement in Northern Ireland. This contributed to the outbreak of the Troubles in the late 1960s, a conflict that would engulf Northern Ireland for the next thirty years. In 1970 he became Member of Parliament for North Antrim and the following year he founded the Democratic Unionist Party (DUP), which he would lead for almost forty years. In 1979 he became a Member of the European Parliament.

In 1951, a congregation of the Presbyterian Church in Ireland (PCI) was forbidden by church authorities to hold a meeting in their own church hall at which Paisley was to be the speaker. In response, the Leaders of that congregation left the PCI and began a new denomination, the Free Presbyterian Church of Ulster, with Paisley, who was just 25 years old at the time. Paisley soon became the leader (or moderator) of the Free Presbyterian Church and was re-elected every year, for the next 57 years.

In 1956, Paisley was one of the founders of Ulster Protestant Action (UPA). Its initial purpose was to organise the defence of Protestant areas against anticipated Irish Republican Army (IRA) activity. It carried out vigilante patrols, made street barricades, and drew up lists of IRA suspects in both Belfast and in rural areas. The UPA was to later become the Protestant Unionist Party in 1966. UPA factory and workplace branches were formed, including one by Paisley in Belfast's Ravenhill area under his direct control. The concern of the UPA increasingly came to focus on the defence of 'Bible Protestantism' and Protestant interests where jobs and housing were concerned. The UPA also campaigned against the allocation of public housing to Catholics.

When Princess Margaret and Queen Mother met Pope John XXIII in 1958, Paisley had condemned them for "committing spiritual fornication and adultery with the Antichrist". When Pope John died in June 1963, Paisley announced to a crowd of followers that "this Romish man of sin is now in Hell!". He organised protests against the lowering of flags on public buildings to mark the Pope's death.

As Paisley came to dominate UPA, he received his first convictions for public order offences. In June 1959, Paisley addressed a UPA rally in the mainly-Protestant Shankill district of Belfast. During the speech he shouted out the addresses of some Catholic-owned homes and businesses in the area. These homes and businesses were then attacked by the crowd; windows were smashed, shops were looted and "Taigs out" painted on the doors.

From the 1960s, one of his main rivals was civil rights leader and co-founder of the nationalist SDLP, John Hume. British Government papers, released in 2002, show that in 1971 Paisley attempted to reach a compromise with the Social Democratic and Labour Party (SDLP). The attempt was made via then British Cabinet Secretary, Sir Burke Trend. The papers show that Paisley had indicated he could "reach an accommodation with Leaders of the Catholic minority, which would provide the basis of a new government in Stormont." It appears that the move was rejected once it became clear to the SDLP that it would have created a very one-sided alliance. Speaking about the deal in 2002 Paisley said:

In 1964, a peaceful civil rights campaign began in Northern Ireland. The civil rights movement sought to end discrimination against Catholics and those of Catholic background by the Protestant and unionist government of Northern Ireland. Paisley instigated and led loyalist opposition to the civil rights movement over the next few years. He also led opposition against Terence O'Neill, Prime Minister of Northern Ireland. Although O'Neill was also unionist, Paisley and his followers saw him as being too 'soft' on the civil rights movement and opposed his policies of reform and reconciliation.

On 6 June 1966, Paisley led a march to the General Assembly of the Presbyterian Church against what he claimed to be its "Romeward trend". The authorities allowed the marchers to go through the Catholic Cromac Square neighbourhood carrying placards with anti-Catholic slogans. Catholic youths attacked the march and clashed with the RUC. Many were injured and cars and businesses were wrecked. Following the riots, Paisley was charged with unlawful assembly and sentenced to three months in prison. The Belfast Telegraph declared that Paisley's organisations "represent a defiance of lawful authority no less serious in essence than that of the IRA". On 22 July 1966, Paisleyites clashed with the RUC outside Crumlin Road Prison, where Paisley was being held. The next day, Protestant mobs several thousand strong "rampaged through the city, smashing windows and trying to damage businesses owned by Catholics". In response, the authorities banned all meetings and marches in Belfast for three months.

On 30 November 1968, hours before a civil rights march in Armagh, Paisley and Ronald Bunting arrived in the town in a convoy of cars. Men armed with nail-studded cudgels emerged from the cars and took over the town centre to prevent the march. The RUC halted the civil rights march, sparking outrage from Activists. On 25 March 1969, Paisley and Bunting were jailed for organising the illegal counter-demonstration. On 6 May, they were released during a general amnesty for people convicted of political offences.

The civil rights campaign, and attacks on it by loyalists and police, culminated in the August 1969 riots. Catholic Irish nationalists clashed with the police and with loyalists, who invaded Catholic neighbourhoods and burned scores of homes and businesses. This led to the deployment of British troops and is seen by many as the beginning of the Troubles. Journalists Patrick Bishop and Eamonn Mallie said of the rioting in Belfast: "Both communities were in the grip of a mounting paranoia about the other's intentions. Catholics were convinced that they were about to become victims of a Protestant pogrom; Protestants that they were on the eve of an IRA insurrection". After the riots, Paisley is reported to have said:

On 16 April 1970, in a by-election to the Northern Ireland Parliament, Paisley, standing on behalf of the Protestant Unionist Party, won the Bannside seat formerly held by Prime Minister Terence O'Neill. Another PUP candidate, william Beattie, won the South Antrim seat. In the 1970 UK general election, Paisley won the North Antrim seat. These elections were "further evidence of the break-up of the unionist block and the unease among a large section of Protestants about the reform measures introduced under Chichester-Clark".

On 30 September 1971, Paisley and Desmond Boal founded the Democratic Unionist Party (DUP).

The Sunningdale Agreement of December 1973 set up a new government for Northern Ireland in which unionists and nationalists would share power. It also proposed the creation of a Council of Ireland, which would facilitate co-ordination and co-operation between the governments of Northern Ireland and the Republic of Ireland. Paisley and other hardline unionists opposed the Agreement. Specifically, they opposed sharing political power with nationalists and saw the Council of Ireland as a step towards a united Ireland.

On 15 May 1974, the UWC called a general strike aimed at bringing down the Agreement and the new government. A co-ordinating committee was set up to help organize the strike. It included Paisley and the other UUUC Leaders, the Leaders of the UWC, and the heads of the loyalist paramilitary groups. Its chairman was Glenn Barr, a high-ranking member of Ulster Vanguard and the UDA. In its first meeting, Barr arrived late and found Paisley sitting at the head of the table. Barr told him "you might be chairman of the Democratic Unionist Party but I'm chairman of the co-ordinating committee, so move over". Paisley moved from the head of the table but carried the chair away with him and the two argued over the chair itself, with Paisley eventually allowed to keep it as he claimed to need a chair with arms due to back pain.

On 3 May 1977, the UUAC organized a general strike. It was seen by the public as "Paisley's strike", due to his prominent role in it. The main aims of the strike were to restore devolved government to Northern Ireland under a system of simple majority (i.e. unionist) rule, and to force the British Government to introduce tougher security measures against the IRA. As in 1974, loyalist paramilitaries tried to enforce the strike by blocking roads, intimidating workers and attacking businesses that refused to co-operate. However, unlike in 1974, many workers refused to join the strike and the security forces were better prepared. The Ulster Service Corps set up roadblocks and carried out patrols in rural areas. Some members carried guns, although these were generally legally-held firearms. During a speech in the House of Commons, Paisley claimed to have taken part in some of these patrols and encouraged his supporters to join the group. On 10 May, Protestant bus driver Harry Bradshaw was shot dead by loyalists for working during the strike, and UDR soldier John Geddis was killed when loyalists bombed a petrol station that had stayed open. That same day, Paisley, Baird and other members of the UUAC were arrested at a roadblock outside Ballymena. Paisley was charged with obstruction of the highway and then released.

Paisley opposed the European Economic Community (EEC), but stood for election to the European Parliament to give a platform to his views and those of his supporters. In June 1979, in the first election to the European Parliament, Paisley won one of the three Northern Ireland seats. He topped the poll, with 29.8% of the first preference votes. On 17 July, Paisley interrupted the opening proceedings of the European Parliament to protest that the Union Jack outside the building was flying upside down. Louise Weiss, who presided over the Parliament, dealt with the interruption swiftly and later said of it that she was used to dealing with "recalcitrant youngsters". On 18 July, Paisley tried to interrupt Jack Lynch—then Irish Prime Minister and President of the European Council—as he was making a speech in the Parliament. Paisley was shouted down by other MEPs.

Paisley was involved in the Drumcree dispute during the late 1980s and 1990s. He supported the right of the Orange Order, a Protestant unionist fraternal organisation, to march through the Catholic part of Portadown. The Catholic residents sought to ban the yearly march from their area, seeing it as sectarian, triumphalist and supremacist. Paisley was a former member of the Orange Order and belonged to a similar Protestant brotherhood: the Apprentice Boys. He also addressed the yearly gathering of the Independent Orange Order.

In December 1981, the State Department of the United States revoked Paisley's visa, citing his "divisive rhetoric" and forcing him to cancel plans for a two-week speaking and fundraising tour in the US. He insisted the cancellation was part of a "conspiracy between the Thatcher Government and the U.S.A. Government to sell out Ulster".

Led by Paisley and UUP leader James Molyneaux, unionists mounted a major protest campaign against the Agreement, dubbed "Ulster Says No". Both unionist parties resigned their seats in the British House of Commons, suspended district council meetings, and supported a campaign of mass civil disobedience. There were strikes and mass protest rallies. On 23 November 1985, more than 100,000 people attended a rally at Belfast City Hall. The rally was addressed by Paisley and Molyneaux. In his address, Paisley famously stated:

On 30 March 1986, a loyalist march was banned from the Catholic district. At midnight, 3,000 loyalists gathered in the town centre. Led by Paisley, they forced their way past police and marched through the Catholic district. Residents claimed that some of the marchers were carrying guns and that police did little to stop the loyalists attacking their homes. This led to severe rioting between residents and the police.

In 1988, having given advance warning of his intentions, Paisley interrupted a speech being delivered by Pope John Paul II in the European Parliament. Paisley shouted "I denounce you as the Antichrist!" and held up a poster reading "Pope John Paul II ANTICHRIST". Other MEPs jeered Paisley, threw papers at him and snatched his poster, but he produced another and continued shouting. He was admonished by Parliamentary President Lord Plumb, who formally excluded him. He was then forcibly removed from the chamber. Paisley claims he was injured by other MEPs—including Otto von Habsburg—who struck him and threw objects at him. Paisley believed the European Union is a part of a conspiracy to create a Roman Catholic superstate controlled by the Vatican. He claimed in an article that the seat no. 666 in the European Parliament is reserved for the Antichrist.

The Free Presbyterian Church is a fundamentalist, evangelical church, requiring strict separation from "any church which has departed from the fundamental doctrines of the Word of God." At the time of the 1991 census, the church had about 12,000 members, less than 1% of the Northern Ireland population.

In July 1995, residents succeeded in stopping the Orange march from entering their area. Thousands of Orangemen and loyalists engaged in a standoff with the police and army at Drumcree Church. Paisley addressed a rally at Drumcree, telling a crowd of thousands:

Paisley's DUP was initially involved in the negotiations under former United States Senator George J. Mitchell that eventually led to the Good Friday Agreement in 1998, but the party withdrew in protest when Sinn Féin was allowed to participate after the Provisional IRA's 1994 ceasefire. Instead, Paisley travelled to Cameroon with the documentary filmmaker Jon Ronson, filming an episode of the television series Witness called "Dr Paisley, I Presume". Paisley and his party opposed the Agreement in the referendum that followed its signing, which saw it approved by over 70% of the voters in Northern Ireland and by over 90% of voters in the Republic of Ireland.

Paisley continued to denounce the Catholic Church and the Pope after the incident. In a television interview for The Unquiet Man, a 2001 documentary on Paisley's life, he expressed his pride at being "the only person to have the courage to denounce the Pope". However, after the death of Pope John Paul in 2005, Paisley expressed sympathy for Catholics, saying "We can understand how Roman Catholics feel at the death of the Pope and we would want in no way to interfere with their expression of sorrow and grief at this time."

Following rumours and a marked change in his appearance, it was confirmed in July 2004 that Paisley had been undergoing tests for an undisclosed illness, and in 2005 Ian Paisley Jr. confirmed that his father had been gravely ill. Paisley himself later admitted that he had "walked in death's Shadow."

Paisley again retained his North Antrim seat in the 2005 UK general election. In 2005, Paisley was made a Privy Counsellor, an appointment traditionally bestowed upon Leaders of political parties in the British Parliament.

In the October 2006 St Andrews Agreement, Paisley and the DUP agreed to new elections, and support for a new executive including Sinn Féin subject to Sinn Féin acceptance of the Police Service of Northern Ireland, the successor to the Royal Ulster Constabulary. This reversed half a century of Paisley's opposition to Sinn Féin, such as his comments four months previously on 12 July in Portrush, following Orange Order parades, when he said, "[Sinn Fein] are not fit to be in partnership with decent people. They are not fit to be in the government of Northern Ireland and it will be over our dead bodies if they ever get there."

In 2007, Paisley was named as "Opposition Parliamentarian of the Year" in The House Magazine Parliamentary Awards and by The Spectator as "Marathon Man of the Year."

Following his January 2008 retirement as leader of the Free Presbyterian Church and pressure from party insiders, on 4 March 2008, Paisley announced that he would stand down as DUP leader and First Minister of Northern Ireland in May 2008. On 17 April, Peter Robinson was elected unopposed as leader of the DUP and succeeded Paisley as First Minister at a special sitting of the assembly on 5 June 2008.

On 18 June 2010, Paisley was created a life peer as Baron Bannside, of North Antrim in the County of Antrim, and he was introduced in the House of Lords on 5 July 2010. Paisley opted not to take the title of "Lord Paisley" as his wife was already in the House as Baroness Paisley and he said that it would have implied she was "sitting not in her own right but as my wife".

In November 2011, Paisley announced to his congregation, which he had led for over 60 years, that he would retire as minister. He delivered his final sermon to a packed attendance at the Martyrs' Memorial Hall on 18 December 2011, and finally retired from his religious ministry at the age of 85, on 27 January 2012.

In February 2012, Paisley was admitted to hospital with heart problems. Jim Flanagan, Editor of the Ballymena Guardian, who spoke to close family friends, said that Paisley had been able to communicate "to some degree" with family members. A year before, he had had a pacemaker fitted due to cardiac arrhythmia, during his time in the House of Lords. In late December 2013, Paisley was once again taken to hospital for "necessary tests". Ian Paisley Jr. emphasised that they were routine.

On 3 December, Paisley claimed that the Third Force had 15,000–20,000 members. James Prior, Secretary of State for Northern Ireland, replied that private armies would not be tolerated.

Paisley died in Belfast on 12 September 2014. He was buried in Ballygowan, County Down on 15 September following a private funeral and a public memorial for 830 invited guests was held in the Ulster Hall on 19 October 2014. A New York Times obituary reported that late in life Paisley had moderated and softened his stances against Roman Catholics but that "the legacies of fighting and religious hatreds remained."