"She’s always been terrible & unfair, just like most of the Fake News reporters, but I’ll still play the game," Trump, 74, said of Ms Welker on Saturday. "The people know," he added.
Mr Trump's tweet also quoted a post by his son Don Jr, in which the latter linked to an article by the right-leaning New York Post, claiming Ms Welker has "deep Democratic ties".
He also criticised Ms Welker during a campaign rally in Wisconsin on Saturday night, telling crowds of supporters she was unfair and unbalanced in her reporting.
The president's criticism came despite him previously congratulating Ms Welker, NBC's White House correspondent, after the network gave her a weekend anchor slot in January this year.
NBC "made a very wise decision" in promoting Ms Welker, Mr Trump said at the time.
Mr Trump also agreed to have Ms Welker moderate the final presidential clash, with his senior campaign manager, Jason Miller telling Fox News last week he thought she was fair and balanced.
Mr Trump's attack on Ms Welker came just a day after it was revealed that more people watched his democratic challenger, Joe Biden during last week's town hall events when the two candidates appeared separately for questioning on their competing views for the country.
Trailing his challenger by a considerable margin in most major polls, this week's final debate in Nashville, Tennessee is key for the president.
With just over two weeks to go until election day, the commander-in-chief is running out of time to win over undecided voters and and reach out beyond his base.
He is also well behind when it comes to female voters, who could be pivotal in deciding the outcome November's election.
Ms Welker, who is highly regarded by her colleagues, was last month honoured by The Washington Women in Journalism Awards as the outstanding broadcast journalist of the year.
She has not responded to the president's comments.