Sensors　based　on　colorimetry,　fluorescence,　and　electrochemistry　have　been　widely　employed　to　detect　acetylcholinesterase　and　its　inhibitors,　however,　there　are　only　a　minority　of　strategies　for　AChE　detection　based　on　photothermal　method.　This　work　reports　a　versatile　dual-mode　colorimetric　and　photothermal　biosensing　platform　for　acetylcholinesterase　(AChE)　detection　and　its　inhibitor　(paraoxon-ethyl,　a　model　of　AChE　inhibitors)　monitor　based　on　Fe–N–C/H2O2/3,3′,5,5′-tetramethylbenzidine　(TMB)　system.　The　Fe–N–C　with　abundant　active　Fe-Nx　sites　shows　outstanding　peroxidase-mimicking　activity　and　can　be　used　to　promote　the　generation　of　•OH　by　H2O2　to　oxidize　TMB.　However,　the　introduction　of　mercapto　molecules　tending　to　coordinate　with　metal　atoms　result　in　the　block　of　action　site　in　Fe–N–C,　thereby　decrease　its　peroxidase-mimetic　activity.　The　designed　biosensor　principle　is　based　on　the　block　of　active　sites　of　Fe–N–C　by　thiocholine　(TCh,　one　kind　of　mercapto　molecules)　that　can　be　produced　by　acetylthiocholine　(ATCh)　in　the　presence　of　AChE.　Under　optimum　conditions,　the　limit　of　detection　(LOD)　for　AChE　activity　is　1.9　mU　mL−1　(colorimetric)　and　2.2　mU　mL−1　(photothermal),　while　for　paraoxon-ethyl　is　0.012　μg　mL−1　(colorimetric)　and　0.013　μg　mL−1　(photothermal),　respectively.　The　assay　we　proposed　not　only　can　be　designed　to　monitor　AChE　detection　and　its　inhibitors,　but　also　can　be　easily　extended　for　the　detection　of　other　biomolecules　relate　to　the　generation　or　consumption　of　H2O2.　©　2022　Elsevier　B.V.