It was his strongest criticism of Judge Tarek Bitar since the investigator's appointment.
“The targeting is clear, you are picking certain officials and certain people. The bias is clear,” he said in televised comments. He said Mr Bitar would never reach the truth if he continued with the investigation.
The Hezbollah Secretary General called for Mr Bitar to be replaced, saying he wanted a judge that was “transparent".
Mr Bitar is not permitted to speak to the media in his capacity as an investigating judge.
The investigation into the explosion on August 4, 2020, one of the biggest non-nuclear blasts in history, has made little headway amid a campaign against Mr Bitar and pushback from powerful Lebanese factions.
The blast killed more than 200 people, injured thousands and destroyed swathes of the capital. But no top official has been held accountable.
Wafik Safa, a senior official in the powerful Iran-backed Hezbollah, warned Mr Bitar last month that the group would remove him from the inquiry, according to a journalist and a judicial source.
Mr Bitar's efforts to question former and current state officials, including the prime minister at the time of the blast, on suspicion of negligence have been repeatedly blocked.
Meanwhile, Lebanese Prime Minister Najib Mikati said in an interview last week that senior government officials must be tried before a special body in line with the constitution. He also said he has signed a draft law to lift immunity from all officials who may be implicated in last year's Beirut port explosion.
Three former ministers charged with criminal negligence in connection with the Beirut port blast have also asked the Lebanon Supreme Court to remove Mr Bitar, citing doubts about his impartiality, the state-run National News Agency reported last week.
The Court of Appeal said it had no authority to rule on requests filed by former ministers Ali Hassan Khalil, Ghazi Zeaiter, and Nohad Mashnouk to replace Mr Bitar.
Mr Bitar has scheduled sessions to question former finance minister Mr Khalil and former public works Mr Zeiter, both allies of Hezbollah, this week. They are not expected to appear.
Although Mr Bitar has sought to question several politicians who are allied to the group, he has not tried to question members of Hezbollah itself.
Mr Bitar is the second judge whose investigation has been stymied by powerful factions in Lebanon, where a lack of high-level accountability is blamed for systemic corruption, governing gridlock and economic meltdown.
His predecessor Fadi Sawan was removed in February by a Lebanese court, after a complaint filed against him by a former official he had charged with negligence.