Appreciating senior Congress leader Mallikarjun Kharge for making the same demand, Ambedkar said, "The Prime Minister should acknowledge that the person whom he has declared as his guru is bent upon creating chaos in this country."
"The prime minister is due for elections by 2019. He will have to answer the question whether he believes in the guru who believes in chaos; therefore I request the prime minister that he should make himself very clear in Lok Sabha by making a statement," he added.
The chief of Bharipa Bahujan Mahasangh party also said the judge to be appointed for the judicial investigation into the matter should not be a Dalit.
Ambedkar also appealed to protesters to maintain calm and agitate peacefully.
The protests were held in Nagpur, Pune and Baramati and incidents of road blockades, arson and stone-pelting were reported in Mumbai and neighbouring areas.
On Wednesday, Ambedkar withdrew the bandh called in Maharashtra to protest the state government's failure in stopping the violence that broke out in Bhima-Koregaon village on Monday.
Maharashtra Chief Minister Devendra Fadnavis has ordered a probe into the death of a youth, who was killed in the clashes.
The chief minister has also announced a compensation of Rs 10 lakh for the victim's kin.
Tension has gripped many parts of the state after clashes broke out between the Dalits and alleged right-wing groups on Monday during the bicentenary celebration of the Bhima-Koregaon battle near Pune.
The matter rose out of the objection by the right-wing groups to the observance of 'Victory Day' since they considered it to be an anti-national celebration.
In the battle, which was fought between the British East India Company, containing Dalits in its infantry, and the Peshwas, who were upper-caste Brahmins, at Koregaon Bhima on January 1, 1818, the Marathas ultimately withdrew. Since then the Dalit communities have been viewing it as a symbolic victory over the upper-caste Brahmins.