The Serbian was sent off for a touchline altercation in the semi-final second leg and his suspension from the clash with Sevilla on Wendesday has now been upheld
The Serbia international was sent off by referee Mark Clattenburg three minutes after being substituted in his side's last-four second leg with Juventus after clashing with Mirko Vucinic on the touchline.
Benfica opted to appeal against the decision in order to ensure Markovic could play in Wednesday's final against Sevilla, but Uefa has rejected the Portuguese champions' plea.
"The Uefa Appeals Body today rejected the appeal of SL Benfica against the red card shown to Lazar Markovic during their Uefa Europa League semi-final second leg versus Juventus on 1 May," the governing body announced.
"Therefore, the Control and Disciplinary Body decision of 6 May is upheld. This verdict is final.
"As a result, the forward is suspended from tonight's final against Sevilla FC to be played at Juventus Stadium."
Benfica will also be without midfielder Enzo Perez and Eduardo Salvio for the clash in Turin after they picked up bans in the semi-final.