Holden took charge at the Valley in December 2022 and guided the club to a 10th-place finish last term despite sitting in 18th upon his arrival.
The Addicks have taken three points from their opening five league games, with their only victory coming against Leyton Orient on the opening day. They were also knocked out of the Carabao Cup on the opening round by Newport.
The London outfit were beaten 2-1 by Oxford at the weekend despite Alfie May’s equaliser just after the hour mark.
Assistant Danny Senda and goalkeeping coach Glyn Shimell have also left their positions, the club has confirmed.
Jason Pearce will take charge of the side on an interim basis while the recruitment process for a new manager is carried out.
“I would like to thank Dean and his staff for their hard work and dedication and sincerely wish them all the best in their future careers.
“Dean’s work in steering the club clear of relegation last season should not be forgotten, nor his development of many younger players.”
Holden began his playing career at Wanderers, making his professional debut under Sam Allardyce.
He went on to have spells with Oldham, Peterborough and Shrewsbury among others before hanging up his boots.
The 43-year-old has also had a spell in the dugout at Bristol City – as well as taking interim charge of Stoke – in recent years.
Charlton are back in action on Saturday afternoon when they host Fleetwood, who lost 1-0 against Shrewsbury over the weekend.