Synthesis　of　lignin-based　carbon　dots　(LCDs)　with　high　quantum　yield　(QY),　stable　fluorescence　properties　and　biocompatibility　has　been　a　challenge.　Here,　we　propose　an　improved　two-step　strategy　for　producing　high-quality　LCDs　from　enzymatic　hydrolysis　lignin　(EHL).　The　p-aminobenzenesulfonic　acid　used　in　the　strategy　not　only　provides　nitrogen　and　sulfur　elements,　but　also　tailors　the　disordered　three-dimensional　structure　of　EHL.　The　successful　co-doping　of　N　and　S　elements　favors　the　reduction　of　the　optical　energy　bandgap　(Eg),　resulting　in　a　high　QY　of　45.05%　for　LCDs.　The　LCDs　exhibited　superior　selectivity　and　sensitivity　for　Fe3+　with　a　limit　of　detection　(LOD)　of　0.15　μM　when　Fe3+　concentration　was　50–500　μM.　In　addition,　LCDs　demonstrated　significant　fluorescence　in　HepG2　cells　and　HepG2　cells　loaded　with　LCDs　at　a　concentration　of　80　μg/mL　showed　good　viability,　suggesting　that　they　are　suitable　for　in　vivo　applications.　The　luminescent　centers　of　LCDs　change　during　pH　regulation　and　thus　show　a　special　visual　response　to　pH　changes,　making　them　have　great　potential　for　detecting　metabolism　in　living　cells.　This　work　provides　a　novel　and　low-cost　method　for　fabricating　sustainable　fluorescent　probes　for　chemical　sensing　and　bioimaging.　©　2023　Elsevier　B.V.