The announcement came after reports Wright had written a $90,000 check to Sen. Mike Duffy for improperly claimed housing expenses, the Canadian Broadcasting Corp. said.
Duffy, who represents Prince Edward Island, resigned from the Conservative caucus Thursday but remains in the Senate as an Independent.
"My actions were intended solely to secure the repayment of funds, which I considered to be in the public interest, and I accept sole responsibility," Wright said in a statement.
"It is with great regret that I have accepted the resignation of Nigel Wright as my chief of staff. I accept that Nigel believed he was acting in the public interest, but I understand the decision he has taken to resign," Harper said in a statement.
Duffy announced last month he had repaid about $90,000 claimed over the last three years for Senate housing and living expenses. External auditors are investigating Duffy and two other Senators.
Ray Novak, Harper's principal secretary, will take over as chief of staff.