Marcelino will have to watch Sunday’s game against La Liga leaders Barcelona from the Mestalla stands.
The 52-year-old was handed his marching orders in the 62nd minute for protesting too much to referee Alejandro Hernandez Hernandez.
The Royal Spanish Football Federation (RFEF) banned him for two matches and although Valencia presented evidence to have the punishment reduced, their appeal failed.
Marcelino will be absent from the dugout on Sunday and for next week’s encounter at Getafe.
A statement from the Competition Commitee of the RFEF read: “Having evaluated the visual and written allegations presented by Valencia CF, we consider that what was written in the game report by the referee is compatible with what occurred.
“Hence, the allegations have been rejected and we confirm the two-match ban to Marcelino Garcia Toral and fine him €600 while the club is fined €700.”
The news is a blow for Valencia, who have impressed under Marcelino since his arrival this summer as a replacement for Voro.