The　abnormal　level　of　alkaline　phosphatase　(ALP)　in　human　blood　stream　indicates　health　problem.　A　novel　fluorescent　sensing　system　based　on　competitive　coordination　with　europium　ions　(Eu3+)　integrated　in　a　non-luminescent　lanthanide-metal　organic　framework　nanosheets　(NO2-Eu-MOF　NS)　was　developed　for　ALP　analysis　in　human　serum.　The　fluorescence　signal　is　turned-on　by　the　antenna　ligand　(1,4-phenylenediphosphonic　acid　(PEPP))　that　competitively　coordinates　with　Eu3+　ions　from　the　nanosheet　to　produce　emissive　species　(Eux(PEPP)y(NO2BDC)z)　in-situ.　4-Nitrophenyl　phosphate　(PNPP)　is　a　substrate　of　ALP　and　specifically　hydrolyzed　into　4-nitrophenol.　PNPP　exhibits　a　strong　absorption　in　the　UV-Vis　region　(250–400　nm),　which　covers　the　excitation　wavelength　(280　nm)　of　Eux(PEPP)y(NO2BDC)z.　Therefore,　the　existing　of　PNPP　causes　Eux(PEPP)y(NO2BDC)z　non-emissive　and,　after　hydrolysis　with　ALP,　Eux(PEPP)y(NO2BDC)z　can　be　excited　and　give　a　detectable　fluorescence　signal.　Based　on　the　fluorescence　change,　quantitative　analysis　of　ALP　activity　is　thus　achievable.　This　biosensing　system　was　demonstrated　in　the　determination　of　ALP　activity　with　good　linear　relationship　in　the　range　of　5–1000　U　L−1.　The　limit　of　detection　was　1.1　U　L−1　(S/N　=　3).　The　result　reveals　that　the　biosensor　exhibits　excellent　sensing　performance　and　anti-interference　ability.　It　could　be　potentially　utilized　for　clinical　routine　analysis　of　ALP　activity　in　human　serum.　©　2023　Elsevier　B.V.