Judge Rana Nisar Ahmed, who is conducting the trial within the high-security Adiala Jail in Rawalpindi for security reasons, rejected the bail applications of former city police chief Saud Aziz and former Superintendent of Police Khurram Shahzad and directed officials to arrest them.
The move came after the Federal Investigation Agency submitted a supplementary chargesheet in which it named the two police officers as accused and sought their arrest to take forward its probe into the assassination.
Prosecutors have raised questions about the alleged failure of the two police officers to provide adequate security to Bhutto during an election rally held in Rawalpindi on Dec 27, 2007, the day she was killed in a gun-and-bomb attack.
They have also questioned Aziz's acceptance of a decision by Bhutto's widower Asif Ali Zardari not to allow an autopsy to be conducted on her body and the hosing down of the crime scene soon after the assassination.
Aziz presented some documents and an audio recording of Zardari saying no autopsy should be conducted on Bhutto's body but these were not accepted by the judge.
Prosecutors told the judge that the FIA's probe could move forward only if the two police officers were arrested.
The anti-terrorism court scheduled the next hearing of the assassination case for Jan 7.
Prosecution officials said that the two police officers would be presented before a magistrate on Dec 23, so that they could be remanded to the FIA's custody.
The anti-terrorism court is conducting the trial of five suspects linked to the Pakistani Taliban who have been charged with involvement in Bhutto's assassination.
The report of a UN commission that probed the assassination said Bhuttos killing could have been prevented.
The report criticised the police's decision to hose down the crime scene, and said its failure to collect and preserve evidence "inflicted irreparable damage to the investigation."