Born in 1969, in Woodland Hills, California, Moloney is the niece of Christine Ebersole, and attended an actors' training program at SUNY Purchase. Her early work included television guest roles on ER, Sports Night and Murder, She Wrote.

In 1999, Janel Moloney became a recurring guest star on The West Wing as Donna Moss, assistant to Deputy White House Chief of Staff Josh Lyman (Bradley Whitford). Moloney was originally intended only to be a guest star, like the many other recurring assistants on the show, but the cast and creator Aaron Sorkin—and the fans—quickly noticed the chemistry between Donna and the rest of the cast, particularly Josh. (And in fact she appeared on every episode in the first season as a "guest".) Beginning with the second season premiere, Moloney was listed in the main credits. Her character eventually advanced from Assistant, to campaign advisor to the Vice President of the United States, to Press Secretary for Presidential nominee Matthew Santos and finally to Chief of Staff for the incoming First Lady. The series ended in May 2006. Moloney has received two Emmy nominations, one in 2002 and one in 2004, for Outstanding Supporting Actress in a Drama.

Moloney has also appeared in the TV film Bang, Bang, You're Dead with Thomas Cavanagh and Randy Harrison, and played the part of Amber Frey in Amber Frey: Witness for the Prosecution, broadcast on CBS May 25, 2005.

She currently lives across the street from The West Wing co-star Allison Janney.

During the 2004 U.S. Presidential campaign, Moloney was an active supporter of John Kerry and made public appearances on behalf of his campaign.