Alkaline　phosphatase　(ALP)　plays　an　important　role　in　phosphate　metabolism　processes;　deviation　from　its　normal　level　may　indicate　different　kinds　of　diseases,　so　it　is　highly　necessary　to　develop　some　simple　and　sensitive　methods　to　monitor　the　ALP　level.　In　this　study,　a　simple,　high　selective,　and　sensitive　fluorescent　biosensor　has　been　proposed　for　ALP　activity　determination.　The　Cu(II)-dependent　DNAzyme　(Cu-Enzyme)　are　divided　into　two　parts:　Cu-Enzyme　1　and　Cu-Enzyme　2,　and　labelled　with　alkyne　and　azido　groups,　respectively.　The　Cu-substrate　(Cu-Sub)　is　labelled　with　a　FAM　fluorophore　(6-carboxyfluorescein)　at　the　3＇-end　and　an　additional　quencher　(BHQ1)　at　the　5＇-end.　The　5＇-end　of　Cu-Enzyme　1　is　labelled　with　BHQ1　as　well.　The　hybridization　of　the　Cu-Enzyme　1　and　Cu-Enzyme　2　with　Cu-Sub　strand　results　in　the　low　background　fluorescence　signal　because　the　fluorescence　from　FAM　is　quenched.　The　addition　of　ALP　can　hydrolyze　AA-P　into　AA,　which　can　reduce　Cu(II)　into　Cu(I)　and　in　turn　catalyze　the　cycloaddition　of　Cu-Enzyme　1　and　Cu-Enzyme　2　to　form　a　modified　Cu-Enzyme.　Then　the　modified　Cu-Enzyme　catalyzes　the　cleavage　of　the　Cu-Sub　strands　into　two　pieces.　One　piece　containing　FAM　fluorophore　can　easily　diffuse　into　solution　and　give　off　a　strong　fluorescence　signal.　The　enhanced　fluorescent　intensity　has　a　linear　relationship　with　the　ALP　concentration　in　the　range　of　0.36-54.55　U　L-1　with　the　detection　limit　of　0.14　U　L-1　(S/N　=　3).　The　proposed　biosensor　has　been　successfully　applied　to　detect　ALP　in　serum　samples　with　satisfied　results.　(C)　2016　Elsevier　B.V.　All　rights　reserved.