The objective is to develop a novel method for T4 polynucleotide kinase (T4 PNK) activity determination based on functional nucleic acid fluorescent probes. The experiment designed a DNA probe (PNK-Tb) that could form a hairpin structure. By introducing T4 PNK, ATP and lambda exonuclease (λ exo), the hairpin structure was opened and the G-rich sequences were released. Then, the released G-rich sequences combined with Tb3+ to form G-quadruplex structure, producing a significant fluorescence signal. Through the change of fluorescence signal, T4 PNK could be detected with high sensitivity. The results showed that a label-free DNA fluorescent probe was successfully prepared and Tb3+ was innovatively applied to T4 PNK assay. The linear range of this method was 0~100 U/mL, and the detection limit was 2 U/mL. This strategy has good specificity and can be used to evaluate the inhibitory effect of ADP on T4 PNK activity. In conclusion, the novel strategy for T4 PNK assay based on label-free DNA fluorescence probe is rapid (less than 60 min), with low cost and high sensitivity, and has broad application prospects in drug development and biochemical research.