Ma and his parents originate from Hunan Province in the Republic of China, with Hakka ancestry, but their ancestral home was in Fufeng, Shaanxi Province. His ancestors had migrated from Shaanxi to Jiangxi and then finally to Hunan. He is a descendant of the Three Kingdoms era general Ma Chao, who was from Fufeng, Shaanxi. Researchers had purportedly visited the old residence of Ma's father, Ma Ho-ling, in Kaiyun Town, Hengshan County, Hunan, where they discovered a genealogy book stating that Ma descended from Ma Chao. His mother was Chin Hou-hsiu (1922-2014). Ma was raised Catholic.

Chai claimed that since Ma was born after 1949 and in Shenzhen, he is legally a citizen of the People's Republic of China. Presidential Spokesperson Wang Yu-chi (王郁琦) responded to Chai's charges by reaffirming that all information from the President's Office regarding the President's birth is accurate. Wang also informed that Ma, on his 11 December visit to Hong Kong, was able to obtain records of his birth at Kowloon's Kwong-Wah Hospital and Ma also keeps the original of his birth certificate issued by the Registrar General of Hong Kong, thereby confirming once again his birth in the former British colony instead of mainland China. Copies of Ma's birth certificate have also been previously shown to the public. Wang also dispelled rumors that Ma had received affirmative action in his applications to Jianguo High School and the National Taiwan University with an "overseas Chinese" status. For that, neither President Ma nor his critics was able to provide a definitive proof. The issue of affirmative action remains open.

He was born in Kwong Wah Hospital in Kowloon, Hong Kong on 13 July 1950. His parents were in Hong Kong on the way from Hunan Province of Communist China to Nationalist-held Taiwan after the Chinese Civil War. In a family of five children, Ma was the fourth child and the only son.

Ma earned his LL.B. degree from National Taiwan University in 1972. He served his compulsory military duty in the ROC Marine Corps and Navy from 1972 to 1974, obtaining the rank of lieutenant commander. He then pursued advanced studies in the United States, first earning an LL.M. degree from New York University Law School in 1976 and then an S.J.D. degree from Harvard Law School in 1981. An edited version of his thesis on the Senkaku Islands dispute was published in 1984.

A week before the presidential election, incumbent President Chen Shui-bian vowed to quit if Ma could provide legal documents of the invalidation of his green card. The DPP presidential candidate Frank Hsieh also said that he was willing to withdraw from the race if Ma could prove, using official documents, that his green card was invalidated twenty years ago. Ma responded the next day to the President that he should work on improving Taiwan's economy instead of caring about the election so much; earlier, Ma also provided copies of US non-immigrant visas issued to him during the 1980s and 1990s, claiming the card was invalid, as such visas are not issued to green card holders.

Ma is married to Christine Chow, and the couple has two daughters. Lesley (Ma Wei-chung, 馬唯中) was born in 1981 in New York City while Ma was attending Harvard. She completed her undergraduate studies in life sciences at Harvard University and then her graduate studies at New York University. Ma's younger daughter is Kelly (Ma Yuan-chung, 馬元中), who was born in Taiwan and completed her undergraduate studies at Brown University in Rhode Island.

Ma was deputy secretary-general of the KMT from 1984 to 1988, also serving for a period as deputy of the Mainland Affairs Council (MAC), a cabinet-level body in charge of cross-strait relations. President Lee Teng-hui appointed him ROC Justice Minister in 1993. Ma was relieved of his post in 1996. His supporters claim that firing was caused by his efforts at fighting corruption among politicians and the police. He remained a supporter of the Kuomintang, rather than supporting the New Party formed by KMT supporters who campaigned on an anti-corruption platform. Ma returned to academia and most people at the time believed his political career to have effectively ended.

Ma has received criticism from the DPP, the opposition party, for allegedly praising the PRC on human rights, especially during the 20th anniversary of the Tiananmen Square Protests. Departing from his usual critical view of the Communists' handling of the 1989 protests, Ma made a statement about the PRC's recent improvements in human rights. That same day, he also asked the PRC government to face its history directly and honestly.

In 1998, the KMT fielded Ma to challenge the then-incumbent Taipei mayor Chen Shui-bian of the opposition Democratic Progressive Party (DPP), who was seeking re-election. Despite Chen's public approval rating of around 70%, he was defeated.

Ma's vice-presidential running mate was former premier Vincent Siew, Lien Chan's running mate in the 2000 presidential election.

Ma was able to repair the political damage and, in December 2002, became the leading figure in the KMT by easily winning reelection as mayor of Taipei with the support of 64% of Taipei voters while DPP challenger Lee Ying-yuan received 36%. His solid victory, especially in light of opposition from both President Chen and former President and KMT chairman Lee Teng-hui, led many to speculate about his chances as the KMT candidate for the 2004 presidential elections, although nothing came of it.

During his time as Taipei's mayor, Ma had many conflicts with the central government over matters such as health insurance rates and control of the water supply during the drought. Ma also was implicated in a scandal of Taipei Bank stock releases in 2003. However, the case was dismissed after an investigation by the Taipei prosecutor. He was strongly criticized by the DPP for not allowing the ROC national flag to be flown along with a PRC flag during Asian Women's Football Championship held in Taipei. Ma responded that he was merely following Olympic protocol, which only officially recognizes the Chinese Taipei Olympic Flag and forbids ROC national flags from being shown in an Olympic Game Stadium.

His initiatives in administering the city of Taipei include changing the transliterations of street names and the line and stations of the Taipei Metro to Hanyu Pinyin, as opposed to Tongyong Pinyin. Ma has expressed mild support for Chinese reunification and opposition to Taiwan independence. He opposed the 2004 referendum, which had been widely criticized by the U.S. and PRC. Nevertheless, his opposition to the Anti-Secession Law of the People's Republic of China, while other Leaders of his party remained silent on the issue, led to him being banned from visiting Hong Kong to make a public speaking tour in 2005. He also criticized the PRC for the Tian'anmen crackdown.

Led by Ma Ying-jeou, the Kuomintang made a resounding win in the three-in-one election held on 3 December 2005. The KMT gained six more seats in the mayoral/magistratical race, from eight seats in the last election, to a total of fourteen seats. Before the election, Ma swore that he would quit the chairmanship if his party could not win over half of the seats, which was a first for a KMT chairman. It was a decisive win for Ma Ying-jeou as well, since he took over the party chairmanship only 110 days before. In the election, the KMT won back the counties of Taipei and Yilan, and the city of Chiayi, which had been the Democratic Progressive Party (DPP)'s strongholds for over twenty years. It was the first time in many years that the KMT regained popularity as far south as Cho-Shui River (Zhuo-Shui River).

Ma clarified later that the current KMT policy of retaining the status quo has not changed and has reiterated this position several times; further, he has also reiterated his party's support of the one-China policy. Ma has defined the status quo as the "Five No's." During a visit to the United States in March 2006, he proposed a "proactive" approach to cross-strait relations which he called the "Five Do's."

Ma visited India and Singapore in June 2007 to increase bilateral exchanges as well as to gain legitimacy and experience for his 2008 presidential bid.

On 2 September 2008, Ma declared that the relations between Taiwan and mainland China were a "special relationship not between two nations", but one that can be handled invoking the "1992 consensus" between the two sides. While the governing authorities on mainland China and Taiwan cannot recognise each other as a legitimate government due to legal and constitutional reasons, Ma seeks that they would refrain from denying the other side being the de facto governing authority of one area of the state. On 18 October 2008, Ma said he hoped that a cross-strait peace accord could be reached during his term in office.

In 2009, Ma spoke at a leadership conference in Taipei and called for peace with Beijing and for Taiwan's greater participation in international affairs. He said: "The Chinese civil war of the 1940s must never happen again. Peace never comes easily, because over 1,000 missiles deployed by Beijing are still aimed at Taiwan." In 2014, Ma received the Eisenhower Medallion from People to People International for his efforts in the East China Sea Peace Initiative, set up to mediate territorial disputes.

Taiwan's economy has experienced significant growth since then, growing 10% in 2010, due to strong demand and tourism from mainland China. A framework was signed in 2010 called the Economic Cooperation Framework Agreement (ECFA), which was accompanied by a debate and a protest.

In June 2011, when incumbent Vice President Vincent Siew announced he will retire and not seek a second term, Premier Wu Den-yih was chosen to replace Siew on the KMT's 2012 ticket. Ma was re-elected President with 51.6% of the vote against Democratic Progressive Party chairwoman Tsai Ing-wen.

During a campaigning event in an aboriginal community, Ma made a controversial remark. Responding to a question from an aboriginal woman, Ma said, "If you come into the city, you are a Taipei citizen... Aborigines should adjust their mentality – if you come into the city you have to play by its rules." This statement was thought to be extremely inappropriate.

Between 18 March to 10 April 2014, the Sunflower Student Movement, initiated by a coalition of students and civic groups in the Legislative Yuan and later also the Executive Yuan, was a student-mainly mass movement to protest Ma's trade in services policy with mainland China. On 26 September 2014, a student protester hurled the book Formosa Betrayed at Ma and hit the President, who was not hurt by the incident. The Presidential Office condemned the act of violence.

On 7 November 2015, Ma met and shook hands with Communist Party General Secretary Xi Jinping in Singapore within their capacity as Leader of Taiwan and Leader of Mainland China respectively. The meeting marked the first ever meeting between Leaders of both sides since the end of Chinese Civil War in 1949.

On 11 October 2016, Ma's office announced that he had accepted an invitation to attend and become one of the speakers at the World Chinese Economic Summit in Malacca, Malaysia which would take place on 16–17 November 2016.

In October 2017, a court ruled in Ma's favor in a defamation case he had brought against Liang Wen-chieh (梁文傑) of the Democratic Progressive Party in 2011, after Liang had claimed on TV that Ma had received NT$300 million in illegal political donations during a meeting with Chen Ying-chu (陳盈助). The court ordered Liang to pay NT$1.2 million (US$39,643).