Dulles attended Princeton University and graduated as a member of Phi Beta Kappa in 1908. At Princeton, Dulles competed on the American Whig-Cliosophic Society debate team. He then attended the George Washington University Law School in Washington, D.C.

On June 26, 1912, Dulles married Janet Pomeroy Avery (1891–1969), a first cousin of David Rockefeller. They had two sons and a daughter. Their older son John W. F. Dulles (1913–2008) was a professor of history and specialist in Brazil at the University of Texas at Austin. Their daughter Lillias Dulles Hinshaw (1914–1987) became a Presbyterian minister. Their son Avery Dulles (1918–2008) converted to Roman Catholicism, entered the Jesuit order, and became the first American theologian to be appointed a Cardinal.

In 1915, Dulles's uncle, Robert Lansing, the then-Secretary of State, recruited him to travel to Nicaragua, Costa Rica and Panama, ostensibly on Sullivan & Cromwell company Business, but in reality to sound out Latin American heads of state on aiding the US war effort against Germany. Dulles advised Washington to support Costa Rica's dictator, Federico Tinoco, on the grounds that he was anti-German, and also encouraged Nicaragua's dictator, Emianiano Camorro, to issue a proclamation suspending diplomatic relations with Germany. In Panama, Dulles offered waiver of the tax imposed by the United States on the annual Canal fee, in exchange for a Panamanian declaration of war on Germany.

In 1918, President Woodrow Wilson appointed Dulles as legal counsel to the United States delegation to the Versailles Peace Conference where he served under his uncle, Secretary of State Robert Lansing. Dulles made an early impression as a junior diplomat. While some recollections indicate he clearly and forcefully argued against imposing crushing reparations on Germany, other recollections indicate he ensured Germany's reparation payments would extend for decades as perceived leverage militating against Future German borne hostilities. Afterwards, he served as a member of the War Reparations Committee at Wilson's request. He was also an early member, along with Future First Lady Eleanor Roosevelt, of the League of Free Nations Association, founded in 1918 and after 1923 known as the Foreign Policy Association, which supported American membership in the League of Nations.

Dulles, a deeply religious man, attended numerous international conferences of churchmen during the 1920s and 1930s. In 1924, he was the defense counsel in the church trial of Rev. Harry Emerson Fosdick, who had been charged with heresy by opponents in his denomination (the event which sparked the continuing Fundamentalist–Modernist Controversy in the international Christian Churches over the literal interpretation of Scripture versus the newly developed "Historical-Critical" method including recent scientific and archeological discoveries). The case settled when Fosdick, a liberal Baptist, resigned his pulpit in the Presbyterian Church congregation, which he had never joined.

After the Wall Street Crash of 1929, Dulles's previous practice brokering and documenting international loans ended. After 1931 Germany stopped making some of its scheduled payments. In 1934 Germany unilaterally stopped payments on private debts of the sort that Dulles was handling In 1935, with the Nazis in power, Sullivan & Cromwell's junior partners forced Dulles to cut all Business ties with Germany. Dulles was then prominent in the religious peace movement and an isolationist, but the junior partners were led by his brother Allen, so he reluctantly acceded to their wishes.

Dulles served as the Chairman and Co-founder of the Commission on a Just and Durable Peace of the Federal Council of Churches of Christ in America (later the National Council of Churches), the Chairman of the Board for the Carnegie Endowment for International Peace, and a Trustee of the Rockefeller Foundation from 1935 to 1952. Dulles was also a founding member of Foreign Policy Association and Council of Foreign Relations.

In the late 1940s, as a general conceptual framework for contending with world communism, Dulles developed the policy known as rollback to serve as the Republican Party's alternative to the Democrats' containment model. It proposed taking the offensive to push Communism back rather than defensively containing it within its areas of control and influence.

Dulles was a prominent Republican and a close associate of Gov. Thomas E. Dewey of New York, who became the Republican presidential nominee in the elections of 1944 and 1948. During the 1944 and 1948 campaigns Dulles served as Dewey's chief foreign policy adviser. In 1944, Dulles took an active role in establishing the Republican plank calling for the establishment of a Jewish commonwealth in Palestine.

In 1945, Dulles participated in the San Francisco Conference as an adviser to Arthur H. Vandenberg and helped draft the preamble to the United Nations Charter. He attended the United Nations General Assembly as a United States delegate in 1946, 1947 and 1950.

Governor Dewey appointed Dulles to the United States Senate to fill the vacancy caused by the resignation of Democratic incumbent Sen. Robert F. Wagner, who resigned due to ill health. Dulles served from July 7 to November 8, 1949. He lost the 1949 special election to finish the term to Democratic nominee Herbert H. Lehman.

The singer Carol Burnett rose to prominence in the 1950s singing a novelty song, "I Made a Fool of Myself Over John Foster Dulles". When asked about the song on Meet the Press, Dulles responded with good humor: “I never discuss matters of the heart in public.”

After Eisenhower won the 1952 presidential election, he chose Dulles as Secretary of State. As Secretary of State, Dulles concentrated on building and strengthening Cold War alliances, most prominently the North Atlantic Treaty Organization. He was the Architect of the Southeast Asia Treaty Organization, an anti-Communist defensive alliance between the United States and several nations in and near Southeast Asia. He also helped instigate the 1953 Iranian coup d'état and the 1954 Guatemalan coup d'état. He favored a strategy of massive retaliation in response to Soviet aggression. He advocated support of the French in their war against the Viet Minh in Indochina but rejected the Geneva Accords that France and the communists agreed to, and instead supported South Vietnam after the Geneva Conference in 1954. Suffering from colon cancer, Dulles resigned from office in 1959 and died later that year.

Dulles strongly opposed communism, believing it was "Godless terrorism". One of his first major policy shifts towards a more aggressive position against communism occurred in March 1953, when Dulles supported Eisenhower's decision to direct the Central Intelligence Agency (CIA), then headed by his brother Allen Dulles, to draft plans to overthrow the Prime Minister Mohammed Mossadegh of Iran. This led directly to the coup d'état via Operation Ajax in support of Mohammad Reza Pahlavi, who became the Shah of Iran.

Dulles was named Time's Man of the Year for 1954.

Dulles was one of the pioneers of massive retaliation and brinkmanship. In an article written for Life magazine, Dulles defined his policy of brinkmanship: "The ability to get to the verge without getting into the war is the necessary art." Dulles' hard line alienated many Leaders of nonaligned countries when on June 9, 1955, he argued in a speech that "neutrality has increasingly become obsolete and, except under very exceptional circumstances, it is an immoral and shortsighted conception." Throughout the 1950s Dulles was in frequent conflict with those non-aligned statesmen he deemed excessively sympathetic to Communism, including India's V.K. Krishna Menon.

In November 1956, Dulles strongly opposed the Anglo-French invasion of the Suez Canal zone in response to the Suez Crisis. During the most crucial days he was hospitalized after surgery and did not participate in Washington's decision-making. However, by 1958 Dulles had become an outspoken opponent of President Gamal Abdel Nasser of Egypt and prevented him from receiving arms from the United States. This policy allowed the Soviet Union to gain influence in Egypt.

This quote is sometimes attributed to Dulles: "The United States of America does not have friends; it has interests." The words were spoken by President Charles de Gaulle of France, but when Dulles traveled to Mexico in 1958, anti-American protesters held up signs reading "The U.S. has no friends, only interests."

Dulles was posthumously awarded the Medal of Freedom and the Sylvanus Thayer Award in 1959. A central West Berlin road was named John-Foster-Dulles-Allee in 1959 with a ceremony attended by Christian Herter, Dulles' successor as Secretary of State.

The Washington Dulles International Airport in Dulles, Virginia and John Foster Dulles High, Middle, and Elementary Schools in Sugar Land, Texas (including the street (Dulles Avenue) where the school campuses are located), were named in his honor, as is John Foster Dulles Elementary School in Cincinnati, Ohio. New York named the Dulles State Office Building in Watertown, New York in his honor. In 1960 the U.S. Post Office Department issued a commemorative stamp honoring Dulles.