The order marked a significant escalation in Trump's targeting of political adversaries and could lay the groundwork for arguments by the Republican that a range of Biden's actions as president were invalid.
Biden responded in a statement on Wednesday night: "Let me be clear: I made the decisions during my presidency. I made the decisions about the pardons, executive orders, legislation, and proclamations. Any suggestion that I didn't is ridiculous and false."
The Justice Department under Democratic and Republican administrations has recognised the use of an autopen to sign legislation and issue pardons for decades, Trump presented no evidence that Biden was unaware of the actions taken in his name, and the president's absolute pardon power is enshrined in the US Constitution.
"This conspiracy marks one of the most dangerous and concerning scandals in American history," Trump wrote in a memo. "The American public was purposefully shielded from discovering who wielded the executive power, all while Biden's signature was deployed across thousands of documents to effect radical policy shifts."
Trump directed Attorney General Pam Bondi and White House counsel David Warrington to handle the investigation. Representatives for Biden did not immediately respond to a request for comment.
It's unclear how far Trump will push this effort, which would face certain legal challenges. But it reflects his fixation on Biden, who defeated him in 2020, an election that Trump never conceded and continues to falsely claim was rigged against him.
Biden issued pardons for his two brothers and his sister shortly before leaving office, hoping to shield them from potential prosecution under Trump, who had promised retribution during last year's campaign. Other pardon recipients included members of a congressional committee that investigated the January 6, 2021, riot at the US Capitol.
House Oversight Chairman James Comer of Kentucky, a Republican, requested transcribed interviews with five Biden aides, alleging they had participated in a "cover-up" that amounted to "one of the greatest scandals in our nation's history."
"These five former senior advisers were eyewitnesses to President Biden's condition and operations within the Biden White House," Comer said in a statement. "They must appear before the House Oversight Committee and provide truthful answers about President Biden's cognitive state and who was calling the shots."
Democrats have dismissed the effort as a distraction.
Comer cited the book "Original Sin" by CNN's Jake Tapper and Axios' Alex Thompson, which details concerns and debates inside the White House and Democratic Party over Biden's mental state and age.
In the book, Tapper and Thompson wrote, "Five people were running the country, and Joe Biden was at best a senior member of the board."
Biden and members of his family have vigorously denied the book's claims.
"This book is political fairy smut for the permanent, professional chattering class," said Naomi Biden, the former president's granddaughter.
Biden withdrew from the presidential race last summer after a debate against Trump in which he appeared to lose his train of thought multiple times, muttered inaudible answers and misnamed different government programs.
He was replaced on the ticket by Kamala Harris, who lost the election to Trump.
