The Republican People’s Party (CHP) accused TRT of “abusing public office” and vowed to take legal action.
The opposition party has previously protested at TRT “bias” towards President Recep Tayyip Erdogan and his Justice and Development Party (AKP).
The election will take place on June 7.
In a statement on the CHP website, deputy leader Bulent Tezcan said: “By taking the decision not the broadcast the advertisement, TRT has created a new scandal.
“The main purpose of state-funded television in all democratic countries is fairness of broadcasting. TRT’s direction is committing the crime of abuse of public office.”
Bulent Tezcan also reminded the state TV that it is “owned by the public”.
TRT has so far declined to comment.
The opposition ad featured the slogan “we applaud as a nation” and criticized the “oppression” of justice, freedom and secularism in Turkey.
It urged voters to attend CHP’s first mass election rally on April 11.
The controversy follows a ruling by a court in Ankara on April 9, which ordered CHP leader Kemal Kilicdaroglu to pay damages for insulting President Recep Tayyip Erdogan in a speech in 2013.
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