An　upconversion　signal　readout　platform　for　the　detection　of　alkaline　phosphatase　(ALP)　was　constructed　by　combining　the　inner　filter　effect　(IFE)　and　the　cascade　signal　amplification　strategy.　First,　3,　3′,　5,　5′　-tetramethylbenzidine　(TMB)　is　oxidized　by　Ag+　to　the　oxidative　product　(TMBox),　which　quenches　the　up-conversion　luminescence　(UCL)　of　the　ligand-free　up-conversion　nanoparticles　(UCNPs)　through　the　IFE　principle.　However,　when　in　the　presence　of　ascorbic　acid　2-phosphate　(AAP)　and　ALP,　AAP　is　catalytically　hydrolyzed　by　ALP　to　produce　ascorbic　acid　(AA),　which　could　not　only　decrease　TMBox　formation　by　reducing　Ag+,　but　also　reduce　the　TMBox　generated　to　TMB,　thereby　resulting　in　fluorescence　recovery　of　UCNP.　Under　optimal　experimental　conditions,　ALP　was　linearly　correlated　in　the　range　of　0.15∼8　U/L　with　a　LOD　of　0.032　U/L　(S/N=3).　In　addition,　the　developed　method　was　applied　to　the　detection　of　ALP　in　actual　serum　samples,　and　the　detection　result　was　close　to　that　of　the　traditional　pNPP-based　standard　assay,　which　showed　that　the　UCNP-TMB　nanosystem　has　great　potential　to　accurately　detect　ALP　in　the　clinical　bioassay.　©　2023　Elsevier　B.V.