Development of a highly sensitive and selective detection method for alkaline phosphatase (ALP) is meaningful in food safety and clinic diagnosis. In this study, we have developed a new fluorescent sensor for ALP activity detection with polymer dots (PDs) based on inner filter effect. The high quantum yield PDs are prepared by 1,4-benzoquinone and ethylenediamine via addition reaction and autopolymerization in aqueous phase under mild conditions. This PDs sensor technology is an effective turn-off fluorescence strategy based on inner filter effect between PDs and p-nitrophenol generated from the ALP enzymatic substrate p-nitrophenylphosphate. The fluorescent assay demonstrates a good linear relationship within 0.1-20U/L (R-2=0.998) and a low detection limit of 0.01U/L. The analysis results of serum samples and inhibitor screening assay suggest that our proposed sensing approach can be used for ALP activity detection in biochemical applications and for the screening of ALP inhibitors in drug discovery.