Hamlin was born to Cyrus Hamlin and his wife Anna, née Livermore, in Paris (in modern-day Maine, then a part of Massachusetts). He was a descendant in the sixth generation of English colonist James Hamlin, who had settled in the Massachusetts Bay Colony in 1639. He was a grandnephew of U.S. Senator Samuel Livermore II of New Hampshire.

Hamlin attended the district schools and Hebron Academy and later managed his father's farm. From 1827 to 1830 he published the Oxford Jeffersonian newspaper in partnership with Horatio King.

Hamlin married Sarah Jane Emery of Paris Hill in 1833. Her father was Stephen Emery, who was appointed as Maine's Attorney General in 1839–1840. Hamlin and Sarah had four children together: George, Charles, Cyrus and Sarah.

Hamlin's political career began in 1835, when he was elected to the Maine House of Representatives. Appointed a Major on the staff of Governor John Fairfield, he served with the militia in the bloodless Aroostook War of 1839. He facilitated negotiations between Fairfield and Lieutenant Governor John Harvey of New Brunswick, which helped reduce tensions and make possible the Webster–Ashburton Treaty, which ended the war.

Hannibal's older brother, Elijah Livermore Hamlin, was President of the Mutual Fire Insurance Co. of Bangor, and the Bangor Institution for Savings. He was twice an unsuccessful candidate for Governor of Maine in the late 1840s, though he did serve as Mayor of Bangor in 1851–52. The brothers were members of different political parties (Hannibal a Democrat, and Elijah a Whig) before both becoming Republican in the later 1850s.

Upon returning from Spain, Hamlin retired from public life to his home in Bangor, Maine, which he had purchased in 1851. The Hannibal Hamlin House – as it is known today – is located in central Bangor at 15 5th Street; incorporating Victorian, Italianate, and Mansard-style architecture, the mansion was posted to the National Register of Historic Places in 1979.

Sarah died in 1855. The next year, Hamlin married her half-sister, Ellen Vesta Emery in 1856. They had two children together: Hannibal E. and Frank. Ellen Hamlin died in 1925.

From the very beginning of his Service in Congress, Hamlin was prominent as an opponent of the extension of slavery. He was a conspicuous supporter of the Wilmot Proviso and spoke against the Compromise Measures of 1850. In 1854, Hamlin strongly opposed the passage of the Kansas–Nebraska Act, which repealed the Missouri Compromise. After the Democratic Party endorsed that repeal at the 1856 Democratic National Convention, on June 12, 1856, he withdrew from the Democratic Party and joined the newly organized Republican Party, causing a national sensation.

The Republicans nominated Hamlin for Governor of Maine in the same year. He carried the election by a large majority and was inaugurated on January 8, 1857. In the latter part of February 1857, however, he resigned the governorship. He returned to the United States Senate, serving from 1857 to January 1861.

Beginning in 1860, Hamlin was a member of Company A of the Maine Coast Guard, a militia unit. When the company was called up in the summer of 1864, Hamlin was told that because of his position as Vice President, he did not have to take part in the muster. He opted to serve, arguing that he could set an Example by doing the duty expected of any citizen, and the only concession made because of his office was that he was quartered with the officers. He reported to Fort McClary in July, initially taking part in routine assignments including guard duty, and later taking over as the company cook. He was promoted to corporal during his Service, and mustered out with the rest of his unit in mid-September.

Although Hamlin narrowly missed becoming President, his vice presidency would usher in a half-century of sustained national influence for the Maine Republican Party. In the period 1861–1911, Maine Republicans occupied the offices of Vice President, Secretary of the Treasury (twice), Secretary of State, President pro tempore of the United States Senate, Speaker of the United States House of Representatives (twice), and would field a presidential nominee in James G. Blaine, a level of influence in national politics unmatched by subsequent Maine political delegations.

For the 1864 election, Hamlin was replaced as Vice Presidential nominee by Andrew Johnson, a Southern Democrat chosen for his appeal to Southern Unionists. After leaving the vice presidency, Hamlin served as Collector of the Port of Boston, a lucrative post to which he was appointed by Johnson after the latter succeeded to the presidency following Lincoln's assassination. However, Hamlin later resigned as Collector because of his disagreement with Johnson over Reconstruction of the former Confederacy.

In June 1864, the Republicans and War Democrats joined to form the National Union Party. Although Lincoln was renominated, War Democrat Andrew Johnson of Tennessee was named to replace Hamlin as Lincoln's running mate. Lincoln was seeking to broaden his base of support and was also looking ahead to Southern Reconstruction, at which Johnson had proven himself adept as military governor of occupied Tennessee. Hamlin, by contrast, was an ally of the Northern "Radical Republicans" (who would later impeach Johnson). Lincoln and Johnson were elected in November 1864, and Hamlin's term expired on March 4, 1865.

In 1869, Hamlin was elected again to the U.S. Senate, and he served two terms. After leaving the Senate in 1881, he served briefly as United States Ambassador to Spain before returning to Maine in late 1882. In retirement, Hamlin was a resident of Bangor, Maine, where he died in 1891. He was buried at Mount Hope Cemetery in Bangor.

Hannibal's nephew (Elijah's son) Augustus Choate Hamlin was a physician, Artist, mineralogist, author, and Historian. He was also Mayor of Bangor in 1877–78, and a founding member of the Bangor Historical Society.

Not content with private life, Hamlin returned to the U.S. Senate in 1869 to serve two more 6-year terms before declining to run for re-election in 1880 because of an ailing heart. His last duty as a public servant came in 1881 when Secretary of State James G. Blaine convinced President James A. Garfield to name Hamlin as United States Ambassador to Spain. Hamlin received the appointment on June 30, 1881, and held the post until October 17, 1882.

On Independence Day, July 4, 1891, Hamlin collapsed and fell unconscious while playing cards at the Tarratine Club he founded in downtown Bangor. He was then placed on one of the club's couches and died a few hours later. He was 81. The couch is preserved at the Bangor Public Library. Hannibal Hamlin was buried in the Hamlin family plot at Mount Hope Cemetery in Bangor, Maine.

Hannibal's first cousin Cyrus Hamlin, who was a graduate of the Bangor Theological Seminary, became a missionary in Turkey, where he founded Robert College. He later became President of Middlebury College in Vermont. His son, A. D. F. Hamlin, Hannibal's first cousin once removed, became a professor of architecture at Columbia University and a noted architectural Historian. There are biographies of Hamlin by his grandson Charles E. Hamlin (published 1899, reprinted 1971) and by H. Draper Hunt (published 1969).

Hamlin had four sons who grew to adulthood: Charles Hamlin, Cyrus Hamlin, Hannibal Emery and Frank Hamlin. Charles and Cyrus served in the Union forces during the Civil War, both becoming generals, Charles by brevet. Cyrus was among the first Union officers to argue for the enlistment of black troops, and himself commanded a brigade of freedmen in the Mississippi River campaign. Charles and sister Sarah were present at Ford's Theater the night of Lincoln's assassination. Hannibal Emery Hamlin was Maine Attorney General from 1905 to 1908. Hannibal Hamlin's great-granddaughter Sally Hamlin was a child actor who made many spoken word recordings for the Victor Talking Machine Company in the early years of the 20th century.

There is also a building on the University of Maine Campus, in Orono, named Hannibal Hamlin Hall. This burned down in 1945, in a fire that killed two students, but was subsequently rebuilt. Hannibal Hamlin Memorial Library is next to his birthplace in Paris, Maine.

Hamlin County, South Dakota is named in his honor, as are Hamlin, Kansas; Hamlin, New York; Hamlin, West Virginia; Hamlin Township; Hamlin Lake in Mason County, Michigan; and, Hamlin, a small Maine village that is a U.S.–Canada border crossing with Grand Falls, New Brunswick. There are statues in Hamlin's likeness in the United States Capitol and in a public park (Norumbega Mall) in Bangor, Maine.