James was born Alexander Franklin James in Kearney, Missouri, to Baptist minister Reverend Robert Sallee James and his wife Zerelda (Cole) James, who had moved from Kentucky. He was the oldest of three children. His father died in 1851 and his mother remarried Benjamin Simms in 1852. After his death she married a third time to Dr. Reuben Samuel in 1855 when Frank was 13 years old. As a child, James showed interest in his late father's sizable library, especially the works of william Shakespeare. Census records show that James attended school regularly, and he reportedly wanted to become a Teacher.

After the withdrawal of regular Confederate troops in the fall of 1861, a bitter guerrilla conflict soon began between bands of pro-Confederate irregulars (commonly known as bushwhackers) and the Union homeguards. By early 1863, Frank, ignoring his parole and oath of allegiance, had joined the guerrilla band of Fernando Scott, a former saddler. He soon switched to the more active command led by william Clarke Quantrill.

Union militiamen searching for Fernando Scott raided the Samuel farm and hanged Dr. Reuben Samuel (though not fatally), Frank's stepfather, torturing him to reveal the location of the guerrillas. Shortly afterward, Frank took part with Quantrill's company in the August 21, 1863 Lawrence Massacre where approximately 200 mostly unarmed civilians were killed.

Frank James was paroled July 27, 1865 in Nelson County, Kentucky. There is a report that after his parole, Frank was involved in a gunfight in Brandenburg, Kentucky with four Soldiers that resulted in two Soldiers killed, one wounded, and Frank wounded in the hip. However, there is an alternative account that claims in the autumn of 1865, Frank, who was in Kentucky going to Missouri, was suspected of stealing horses in Ohio and that Frank shot two members of a posse and escaped.

During his years as a bandit, James was involved in at least four robberies between 1868 and 1876 that resulted in the deaths of bank employees or citizens. The most famous incident was the disastrous Northfield, Minnesota, raid on September 7, 1876, that ended with the death or capture of most of the gang.

He was tried for only two of the robberies/murders – one in Gallatin, Missouri for the July 15, 1881 robbery of the Rock Island Line train at Winston, Missouri, in which the train Engineer and a Passenger were killed, and the other in Huntsville, Alabama for the March 11, 1881 robbery of a United States Army Corps of Engineers payroll at Muscle Shoals, Alabama. Among others, former Confederate General Joseph Orville Shelby testified on James' behalf in the Missouri trial. He was acquitted in both Missouri and Alabama. Missouri accepted legal jurisdiction over him for other charges, but they never came to trial. He was never extradited to Minnesota for his connection with the Northfield Raid.

Five months after the killing of his brother Jesse in 1882, Frank James boarded a train to Jefferson City, Missouri, where he had an appointment with the governor in the state capitol. Placing his holster in Governor Crittenden's hands, he explained,

In the last thirty years of his life, James worked a variety of jobs, including as a shoe salesman and then as a Burlesque theater ticket taker in St. Louis. One of the theater's spins to attract patrons was their use of the phrase "Come get your ticket punched by the legendary Frank James." He also served as an AT&T telegraph operator in St. Joseph, Missouri. James took up the lecture circuit, while residing in Sherman, Texas. In 1902, former Missourian Sam Hildreth, a leading thoroughbred horse trainer and owner, hired James as the betting commissioner at the Fair Grounds Race Track in New Orleans. He returned to the North Texas area where he was a shoe salesman at Sanger Brothers in Dallas. The Tacoma Times reported in July, 1914 that he was picking berries at a local ranch there in Washington state and planned to buy a farm nearby. He was also part of a Chicago investment group which purchased the Fletcher Terrell's Buckskin Bill's Wild West Show, third in size after the Buffalo Bill and Pecos Bill shows.

In his final years, James returned to the James Farm, giving tours for the sum of 25 cents. He died there on February 18, 1915, aged 72 years. He left behind his wife Annie Ralston James and one son.