Herein,　a　ratiometric　fluorescence　sensor　based　on　the　fluorescence　resonance　energy　transfer　(FRET)　strategy　was　proposed　to　detect　alkaline　phosphatase　(ALP)　activity　and　its　inhibitor　(sodium　vanadate,　Na3VO4).　The　principle　was　that　blue　fluorescent　nitrogen　and　sulfur　co-doped　carbon　dots　(N/S-CDs)　were　designed　as　energy　donors,　and　ALP　induced　the　formation　of　yellow　fluorescent　resultant　2,3-diaminophenazine　(DPA)　as　an　energy　acceptor,　causing　the　fluorescence　to　change　from　blue　to　yellow.　Indeed,　the　FRET　efficiency　reached　up　to　93.2%,　which　is　higher　than　most　reported　FRET　strategies　for　sensing　and　imaging.　After　the　recognition　of　ALP,　the　fluorescence　intensity　achieved　a　ratiometric　change,　accompanied　by　a　significant　transition　in　fluorescence　color　(Stokes　wavelength　shifted　about　220　nm),　which　can　achieve　quantitative　detection　with　a　limit　of　detection　being　0.396　U/L　and　visual　qualitative　analysis　of　ALP　activity.　Moreover,　dual-emission　self-correction　ability　can　reduce　external　interference　and　improve　the　stability.　The　strong　coordination　ability　between　pyrophosphate　(PPi)　and　Cu2+and　the　efficient　hydrolysis　of　PPi　by　ALP　determined　the　specificity　of　the　biosensor,　and　satisfactory　results　were　obtained　in　the　detection　of　clinical　human　serum　samples.　The　proposed　ratiometric　fluorescence　biosensor　provides　an　alternative　way　for　effective　evaluation　of　ALP-related　diseases,　and　it　can　also　be　used　for　the　assessment　of　Cu2+,　PPi,　and　Na3VO4content　and　the　screening　of　ALP　inhibitors.　©　2021　American　Chemical　Society