Allott, a Spireites player between 2002 and 2007, has put pen to paper on a two-year deal at Saltergate and becomes John Sheridan's second summer signing following the arrival of another old boy Ian Breckin.
The 31-year-old only joined Tranmere this summer, having been brought to Boundary Park by Ronnie Moore but following his dismissal as manager, Allott has been told he does not figure in the plans of new Rovers boss John Barnes.
"Mark is a proven experienced midfielder who we all know so well.
I am delighted that he has become available and that he immediately wanted to join our promotion push here at Chesterfield," said Spireites boss Sheridan.
Allott made 243 appearances and scored 14 goals in his first spell at Saltergate. He previously played under Sheridan at Oldham.