Justice Tsoho is the second judge to do so.
Mr. Kanu is facing treason charges brought by the Nigerian government for leading a separatist group.
He has been in detention since October 2015.
The first judge in the case, Ahmed Mohammed, stepped down in December 2015, after Mr. Kanu said he had no confidence in him.
In a new petition to the National Judicial Council, Mr. Kanu accused Justice Tsoho of “judicial rascality” and called for his investigation by the council.
In the petition made available to journalists in Abuja on Sunday, Mr. Kanu through his lawyer, Ifeanyi Ejiofor, asked the council to investigate Mr. Tsoho for giving parallel judgements on the same request.
Mr. Ejiofor said after Justice Tsoho decided in favour of the defence in an application for the protection of witnesses by the prosecution, and later ruled in favour of the prosecution, without seeking the permission of a higher court.
At the opening of session on Monday, another counsel to Mr. Kanu, Chuks Muoma, said the continuation of the matter by Mr. Tsoho would amount to a great disservice since there was a petition against him, before the NJC.
JusticeTsoho announced he had stepped down, pending the decision of the council.
“Even if I am cleared of any bias by the NJC, I will not continue with this case,” said Justice Tsoho.