As a bifunctional enzyme, T4 polynucleotide kinase phosphatase (T4 PNKP) catalyzes the phosphorylation of 5'-hydroxyl, and also removes the terminal 3'-phosphate group. This is closely related to the restructuring, replication, and damage repair of nucleic acid. In this paper, we describe a new method for the sensitive detection of T4 PNKP activity based on the isothermal EXPonential amplification reaction (EXPAR). T4 PNKP can be linearly assayed in the range from 0.001 to 0.01 U mL-1 with a detection limit of 7.9 × 10-4 U mL-1. Moreover, the method exhibits high specificity and sensitivity and can be applied in the enzyme analysis of complex serum samples. In view of its simplicity and moderate experimental conditions, the method may suitable for use in a commercial kit for the analysis of T4 PNKP activity.