Roofe was sent off during Rangers' controversial 2-0 Europa League defeat to the Czech side back in March after planting his studs into Kolar's face.
The Czech keeper suffered a fractured skull as a result - but Rangers announced they would contest Roofe's ban, claiming the punishment was "severe".
However, in a statement UEFA confirmed the original sanction would stand.
"The appeal lodged by Rangers FC has been dismissed," it read. "Consequently, the UEFA Control, Ethics and Disciplinary Body's decision of 13 April 2021 is confirmed.
"The CEDB had decided to suspend Rangers FC player, Mr. Kemar Roofe, for four UEFA club competition matches for which he would be otherwise eligible, for dangerously assaulting another player."
The former Leeds and Anderlecht ace will now have to sit out as Gerrard's team kick off their qualifying bid.
Steven Gerrard's newly-crowned champions are scheduled to begin their quest to return to the group stages of the continent's premier club competition on August 3 in the third qualifying round.
Roofe's horror challenge was the first flashpoint on a controversial night at Ibrox. Glen Kamara and his team-mates reacted with fury after the Gers midfielder was allegedly racially abused by Slavia defender Ondrej Kudela.
Kudela was hit with a 10-game suspension - ruling him out of this summer's European Championship pending an appeal- while Kamara was also punished with a three-match ban for allegedly assaulting Kudela in the Ibrox tunnel after the game. Rangers have also appealed against that ban.