He faces 95 counts of gross negligence manslaughter following the fatal crush in the Leppings Lane end allocated to Liverpool fans.
The former chief superintendent appeared via videolink at Preston Crown Court on Monday, confirming his name and entering his plea.
England's state prosecutor, the Crown Prosecution Service (CPS), had applied to lift a historical stay -- an order preventing him being tried -- on Duckenfield, which was put in place in 2000.
Former Sheffield Wednesday club secretary Graham Mackrell, 68, also pleaded not guilty to one charge involving the stadium safety certificate and one health and safety charge.
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