The Tigers confirmed that Duffen had stepped down less than 24 hours after they were forced to issue a statement denying that manager Phil Brown had been sacked.
Duffen departs amid concerns over the club's finances and with the team struggling on the field and in the bottom three of the Premier League.
Former chairman Adam Pearson, who Duffen replaced in June 2007, has been strongly linked with a return to the KC Stadium after stepping down from his post with Derby on Wednesday.
Duffen's departure could now increase the pressure on Brown, with the Tigers supremo having been a consistent defender of the under-fire manager.
Totally amicableA statement on the club's official website read: "The club has received a letter of resignation from Paul Duffen, executive chairman of Hull City AFC and Superstadium Management Company Ltd, which has been accepted and is effective immediately.
"Paul's departure is on a totally amicable basis and he leaves with our best wishes as a friend of the club and its directors.
"The club wishes to express its gratitude to Paul for his outstanding contribution as chairman during a period of unprecedented success for Hull City AFC.
"The recruitment process has commenced in respect of the appointment of a new executive chairman and a further statement in relation to this will be issued by the club on Monday, 2nd November."
Duffen oversaw the club's rise to the top flight for the first time in their history, which was achieved via victory in the 2008 Championship play-off final, with Dean Windass scoring the only goal of the game.
The Tigers made an excellent start to life in the Premier League last term after winning promotion, but faded badly in the second half of the season, only avoiding the drop on the final day.
Ridiculous ambitionDuffen issued his own statement, which read: "When I succeeded Adam Pearson as chairman of Hull City Football Club in June 2007 I was very aware of the responsibility to build on his legacy.
"I articulated an intention to take the Tigers to the previously uncharted waters of the Premier League and to the eternal credit of the players and coaching staff, that seemingly ridiculous ambition was achieved 11 months later thanks to a victorious debut appearance at Wembley in the play-off final.
"The start to our maiden top-flight campaign broke all previous records for a newly promoted side and ensured that 2008 will forever be remembered as the most remarkable year in Hull City's history.
"Football is a results business and in the same way that I enjoyed the successes of the previous two seasons, so I must take ultimate responsibility for the disappointments of 2009.
"It has been a tremendous privilege to lead the Tigers on this amazing journey and I pay tribute to all the staff for their hard work and dedication as well as to the fans for their magnificent support. I am proud to have played my part and wish the club every success in the future."