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.
All three politicians said the judge had overstepped his legal authority by seeking to prosecute senior officials who enjoy parliamentary immunity.
The court ordered the three officials to cover legal fees of 800,000 Lebanese pounds ($45 on the black market rate).
Mr Bitar’s summons was also snubbed by former prime minister Hassan Diab and ex-public works minister Youssef Finianos.
The judge issued an arrest warrant for Mr Finianos in September after he failed to appear for questioning.
Mr Finianos later asked the Supreme Court to remove Mr Bitar over questions of his impartiality. The court has yet to rule on the request.
The Supreme Court removed Mr Bitar’s predecessor, Fadi Sawan, after Mr Zeaiter and Mr Khalil, both members of Parliament Speaker Nabih Berri's political bloc, filed appeals on the same grounds.
Parliament had denied Mr Bitar’s request to question senior officials, saying they should instead be prosecuted before a special body that includes MPs and senior judges, as stipulated by the constitution.
The victims' families have since accused politicians of stalling and attempting to block the investigation into the August 2020 explosion that killed at least 214 people and destroyed large parts of the capital.
Court decision gives hope to victims' families
Families of the victims held their monthly sit-in on Monday to mark the anniversary of the blast and demand justice. A few dozen people, mostly dressed in black, gathered at the port.
Protesters held up photos of loved ones who were killed in the explosion and banners that read "We have had enough, we want the truth".
More than a year later, it remains unclear what triggered the explosion of hundreds of tonnes of chemical fertiliser that were stored for over six years at the port. Questions also remain over who owned the ammonium nitrate and why it was stockpiled for so long at such a vital facility.
The court's decision has given hope to the families, they say, that justice may be served.
Sania Matta, who lost her 24-year-old son last August, told The National: "For once we have hope that the truth will be uncovered. God protect Judge Bitar."
Reports have linked the shipment of explosive chemicals to businessmen with ties to Syrian President Bashar Al Assad, an ally of Lebanon's Iran-backed Hezbollah movement.
Hezbollah has repeatedly criticised Mr Bitar and threatened to remove him from the investigation, accusing him of politicising the case.
The explosion, which toppled Mr Diab’s government and left the country without a functioning Cabinet for a year, compounded the effect of Lebanon’s financial crisis, causing billions of dollars in property damage across Beirut.
The international community has urged Lebanese officials to allow a transparent investigation, with the US and France recently voicing concerns over last week’s suspension of the inquiry.
Mariana, 31, lost her sister in the explosion. She regularly participates in sit-ins for the victims.
She said she was hopeful the case will proceed – not only because of Monday's court decision but also because she has faith in the families' perseverance.
"The families of the victims did not give up. We will never give up."