"It's wrong to think of a fair investigation till he (Chouhan) is there as chief minister. If he doesn't resign it will show that he intends to affect the probe," Singh said.
The Congress had called a strike to protest the Vyapam scam, which it claims was successful with no untoward incident being reported from anywhere.
A few other parties also supported the strike across the state.
Shops were closed in the capital Bhopal and adjoining cities on Thursday. Very few public transport buses and auto-rickshaws plied.
A 40-member team under the supervision of CBI Joint Director R.P. Aggarwal initiated its probe into the Vyapam scam on Monday.
Vyapam (Vyavsayik Pariksha Mandal) conducts recruitment and entrance exams not conducted by the Madhya Pradesh Public Service Commission. The Vyapam scam came to light in 2013 and has since assumed mind-boggling proportions, leading to the arrest of nearly 2,100 people. Nearly 40 people linked in some way or the other to the scam have died.