Accurate　monitoring　of　methylglyoxal　(MGO)　at　cell　and　living　level　was　crucial　to　reveal　its　role　in　the　pathogenesis　of　diabetes　since　MGO　was　closely　related　to　diabetes.　Herein,　a　ratiometric　fluorescence　strategy　was　constructed　based　on　the　capture　probe　2,3-diaminonaphthalene　(DAN)　for　the　specific　detection　of　MGO.　Compared　to　the　fluorescent　probes　with　a　single　emission　wavelength,　the　ratiometric　mode　by　monitoring　two　emissions　can　effectively　avoid　the　interference　from　the　biological　background,　and　provided　additional　self-calibration　ability,　which　can　realize　accurate　detection　of　MGO.　The　proposed　method　showed　a　good　linear　relationship　in　the　range　of　0–75　μm　for　MGO　detection,　and　the　limit　of　detection　was　0.33　μm.　DAN　responded　to　MGO　with　good　specificity　and　was　successfully　applied　for　detecting　the　ex　vivo　MGO　level　in　plasma　of　KK−Ay　mice　as　a　type　II　diabetes　model.　Besides,　the　prepared　DAN　test　strip　can　be　visualized　for　rapid　semi-quantitative　analysis　of　MGO　using　the　naked　eye.　Furthermore,　human　skin　fibroblasts　and　HeLa　cells　were　utilized　for　exogenous　MGO　imaging,　and　ex　vivo　MGO　imaging　was　performed　on　tissues　of　KK−Ay　mice.　All　results　indicated　that　the　DAN-based　ratiometric　fluorescence　probe　can　be　used　as　a　potential　method　to　detect　the　level　of　MGO,　thus　enabling　indications　for　the　occurrence　of　diabetes　and　its　complications.　©　2022　The　Authors.　Published　by　Wiley-VCH　GmbH.