Carbon　dots　(CDs)　have　received　considerable　attention　in　many　application　areas　owing　to　their　unique　optical　properties　and　potential　applications;　however,　the　fluorescent　mechanism　is　an　obstacle　to　their　applications.　Herein,　three-color　emissive　CDs　are　prepared　from　single　o-phenylenediamine　(oPD)　by　regulating　the　ratio　of　ethanol　and　dimethylformamide　(DMF).　Fluorescent　mechanism　of　these　CDs　is　proposed　as　molecular　state　fluorescence.　Reaction　intermediates　are　identified　using　liquid　chromatrography–mass　spectroscopy　(LC–MS)　and　1H　nuclear　magnetic　resonance　(NMR)　spectra.　1H-Benzo[d]imidazole　(BI),　2,3-diaminophenazine　(DAP),　and　5,14-dihydroquinoxalino[2,3-b]　phenazine　(DHQP)　are　proposed　to　be　the　fluorophores　of　blue,　green,　and　red　emissive　CDs　by　comparing　their　optical　properties.　As　per　the　LC–MS　and　1H-NMR　analysis,　DHQP　with　red　emission　tends　to　form　from　DAP　and　oPD　in　pure　ethanol.　By　adding　DMF,　BI　formation　is　enhanced　and　DHQP　formation　is　suppressed.　The　prepared　CDs　exhibit　green　emission　with　DAP.　When　the　DMF　amount　is　＞50%,　BI　formation　is　considerably　promoted,　resulting　in　DAP　formation　being　suppressed.　BI　with　blue　emission　then　turns　into　the　fluorophore　of　CDs.　This　result　provides　us　an　improved　understanding　of　the　fluorescent　mechanism　of　oPD-based　CDs,　which　guides　us　in　designing　the　structure　and　optical　properties　of　CDs.　©　2024　Wiley-VCH　GmbH.