A judge at sport's highest court dismissed the Ukrainian club's request to have its expulsion from the tournament for match-fixing frozen while an appeal proceeds.
The verdict means Greek side PAOK Salonika, who lost to Metalist in the third qualifying round, will face Schalke of Germany in a two-legged play-off later this month.
Metalist, last season's Ukrainian league runners-up, were barred by UEFA on Wednesday after CAS upheld a five-year ban for the club's sports director in a Ukrainian league match-fixing case.
CAS said it had considered written submissions from UEFA and PAOK along with "the urgent request for provisional measures filed by FC Metalist Kharkiv".
And it added: "After having considered the parties' submissions, the deputy president of the appeals arbitration division decided to dismiss FC Metalist's request.
"Consequently, the UEFA decision remains in force, which means that FC Metalist Kharkiv is excluded from the 2013/2014 UEFA competitions."
A UEFA statement said: "UEFA has welcomed the Court of Arbitration for Sport's decision to reject the application for provisional measures made by FC Metalist Kharkiv after the club were banned from UEFA competitions."
UEFA's Champions League rules require that clubs not be implicated in fixing any domestic or international matches since April 2007.