"Really, isn't this a guy who came in and said 'I am Barack Obama's wingman'?" Rove said on "The Story with Martha MacCallum" Tuesday.
"He said he's concerned about the appearance of propriety."
Holder told Fox News on Tuesday that Barr "is paying a price" and sacrificing his credibility by spearheading U.S. Attorney John Durham's ongoing probe into possible misconduct by the intelligence community at the outset of the Russia investigation.
"And then to see how the president is now involved to help the attorney general in that effort gives me pause," Holder told Fox News. "I think the attorney general needs to be a little more sensitive to the appearance that gives. You have to not only be substantively neutral -- you have to appear to be neutral when you are the attorney general of the United States. And I fear that he has crossed a political line."
The former senior advisor to former President George W. Bush criticized Holder for dismissing a complaint against the Black Panther Party in 2008 for voter intimidation at the polls. He also criticized Holder as someone with partisan motives.
"Every attorney general I can remember, when they left the office except for Robert Kennedy, sort of melded back into the legal community -- what is this guy doing?" Rove asked.
"This guy is leading a nationwide effort to attack Republican state legislators and Republican redistricting plans in order to elect more Democrats to the Congress."
Rove said Holder had no right to "lecture" anyone.
"This guy has no right whatsoever to lecture us about impartiality particularly on the part of an attorney general," Rove said.
Fox News' Gregg Re contributed to this report.