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A sensitive label-free fluorescence assay for monitoring T4 polynucleotide kinase (T4 PNK) activity and inhibition was developed based on a coupled λ exonuclease cleavage reaction and SYBR Green I. In this assay, a double-stranded DNA (dsDNA) was stained with SYBR Green I and used as a substrate for T4 PNK. After the 5′-hydroxyl termini of the dsDNA was phosphorylated by the T4 PNK, the coupled λ exonuclease began to digest the dsDNA to form mononucletides and single-stranded DNA (ssDNA). At this moment, the fluorescence intensity of the SYBR Green I decreased because of less affinity with ssDNA than dsDNA. The decreasing extent was proportional to the concentration of the T4 PNK. After optimization of the detection conditions, including the concentration of ATP, amount of λ exonuclease and reaction time, the activity of T4 PNK was monitored by the fluorescence measurement, with the limit of detection of 0.11 U mL −1 and good linear correlation between 0.25–1.00 U mL −1 ( R 2 = 0.9896). In this assay, no label was needed for fluorescence detection. Moreover, the inhibition behaviors of the T4 PNK's inhibitors were evaluated by this assay. The result indicated the potential of using this assay for monitoring of the phosphorylation-related process.