The oath of office ceremony took place on the Senate floor in the U.S. Capitol in Washington, D.C.
Fellow Sen. Kirsten Sinema, D-Ariz., held the Bible, which belonged to the grandmother of Kelly's wife, former Rep. Gabrielle Giffords, D-Ariz. Giffords, along with the couple's two daughters, Claudia and Claire, and Kelly's twin brother, fellow astronaut Scott Kelly, also attended.
Shortly after the ceremony, Sinema tweeted her promise to work with her fellow Democrat from Arizona.
"I look forward to partnering with @SenMarkKelly to cut through Washington dysfunction and deliver results for everyday Arizonans -- and I am confident Mark will uphold the Arizona values of seeking common ground and putting country above party," she wrote.
Kelly defeated incumbent Sen. Martha McSally, R-Ariz., in the November election. She was appointed to the seat after McCain died from brain cancer in 2019.
Kelly won the race by fewer than 80,000 votes, or 2.4% of the vote. His win narrows the Republican majority in the Senate to 52-48. With two runoff races scheduled for January in Georgia, the chamber could end up even, with Vice President-elect Kamala Harris breaking any ties.
This is the first time Arizona has had two Democrats in the Senate since Sens. Carl Hayden and Ernest McFarland in the 1950s.
Kelly will complete the remaining two years on McCain's final six-year term in office.
He and Giffords have been working to prevent gun violence since she survived a politically motivated shooting in 2011 that killed six.