DNA phosphorylation, catalyzed by polynucleotide kinase (PNK), plays significant regulatory roles in many biological events. Here, a novel fluorescent nanosensor based on phosphorylation-specific exonuclease reaction and efficient fluorescence quenching of single-stranded DNA (ssDNA) by a WS2 nanosheet has been developed for monitoring the activity of PNK using T4 polynucleotide kinase (T4 PNK) as a model target. The fluorescent dye-labeled double-stranded DNA (dsDNA) remains highly fluorescent when mixed with WS2 nanosheets because of the weak adsorption of dsDNA on WS2 nanosheets. While dsDNA is phosphorylated by T4 PNK, it can be specifically degraded by λ exonuclease, producing ssDNA strongly adsorbed on WS2 nanosheets with greatly quenched fluorescence. Because of the high quenching efficiency of WS2 nanosheets, the developed platform presents excellent performance with a wide linear range, low detection limit and high signal-to-background ratio. Additionally, inhibition effects from adenosine diphosphate, ammonium sulfate, and sodium chloride have been investigated. The method may provide a universal platform for PNK activity monitoring and inhibitor screening in drug discovery and clinic diagnostics.