A　rapid,　label-free,　and　highly　sensitive　fluorescence　biosensing　platform　has　been　fabricated　for　effectively　detecting　acetylcholinesterase　(AChE)　activity　via　fluorescence　resonance　energy　transfer　(FRET)　between　boron　nitride　quantum　dots　(BNQDs)　and　gold　nanoparticles　(AuNPs).　In　this　work,　AChE　hydrolyzed　acetylthiocholine　(ATCh)　to　generate　thiocholine　(TCh),　which　can　reduce　chloroauric　acid　(HAuCl4)　into　AuNPs　and　in　situ　formation　of　TCh-BNQDs/AuNPs　aggregates,　resulting　in　fluorescence　quenching　of　BNQDs.　Moreover,　paraoxon,　a　organophosphorus　pesticide,　was　used　to　lower　the　activity　of　AChE　and　hinder　the　enzymatic　hydrolysis　reaction,　hence　weakened　the　fluorescence　quenching　of　BNQDs.　Based　on　these　findings,　a　simple　＇one-pot＇　FRET-based　biosensor　has　been　constructed　to　assess　AChE　activity　and　its　inhibitor　by　measuring　the　fluorescence　intensity　of　BNQDs.　The　linear　range　toward　AChE　was　from　0.05　to　6.0　mU/mL　with　the　lower　detection　limit　of　0.0212　mU/mL,　indicating　the　biosensor　out-performs　most　of　the　reported　sensors.　Since　the　activity　of　AChE　has　been　connected　to　a　number　of　diseases,　this　convenient　＇mix　and　detect＇　approach　has　potentially　extensive　application　in　clinic　diagnosis.　On　the　other　hand,　the　inhibition　effect　by　paraoxon　was　evaluated,　and　the　corresponding　half　maximal　inhibitory　concentration　(IC50)　toward　AChE　was　estimated　to　be　2.987　mu　g/L.　As　far　as　we　known,　the　proposed　biosensing　platform　based　on　TCh-BNQDs/AuNPs　nanocomposites　is　the　first　time　to　demonstrate　the　application　of　BNQDs　for　fluormetric　assays　of　enzyme　activity　and　inhibiting　effect.　It　also　opens　up　a　novel　pathway　of　using　BNQDs　to　develop　optical　biosensors　for　analyzing　many　other　analytes.