Hichilema was arrested in April for allegedly failing to give way to President Edgar Lungu's motorcade and has so far been held in detention for nearly two months.
The incident led to the leader of the United Party for National Development being accused of endangering Lungu's life and treason charges followed.
"I am in court because of hatred. This has everything to do with hatred," Hichilema told journalists inside the courtroom.
His lawyers asked the court to throw out the treason charge, arguing it was baseless, but the magistrate referred the case to the High Court with the date for the hearing yet to be announced.
Treason suspects are not eligible for bail in Zambia and, if found guilty, Hichilema could face a possible death sentence.
Hichilema said he hoped the trial would start soon and criticised the police, accusing them of failing to properly investigate the case.
"We need a dedicated judge to deal with our matter expeditiously," he said.
Zambia is considered one of the most stable countries in Africa and Hichilema's arrest and detention has been widely criticised by human rights groups.
In a show of support, the leader of South Africa's main opposition party, Mmusi Maimane, also tried to visit Hichilema in Zambia last month but was barred from entering the country.
The treason case comes after Hichilema made a fifth unsuccessful bid for the presidency last year.
He refused to recognise Lungu as president and has challenged the narrow poll defeat in court.
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