Although　organic　artificial　enzymes　have　been　reported　as　biomimetic　oxidation　catalysts　and　are　widely　used　for　colorimetric　biosensors,　developing　organic　artificial　enzymes　with　high　enzymatic　activity　is　still　a　challenge.　Two-dimensional　(2D)　covalent　organic　frameworks　(COFs)　have　shown　superior　potential　in　biocatalysts　because　of　their　periodic　π-πarrays,　tunable　pore　size　and　structure,　large　surface　area,　and　thermal　stability.　The　interconnection　of　electron　acceptor　and　donor　building　blocks　in　the　2D　conjugated　COF　skeleton　can　lead　to　narrower　band　gaps　and　efficient　charge　separation　and　transportation　and　thus　is　helpful　to　improve　catalytic　activity.　Herein,　a　donor-acceptor　2D　COF　was　synthesized　using　tetrakis(4-aminophenyl)pyrene　(Py)　as　an　electron　donor　and　thieno[3,2-b]thiophene-2,5-dicarbaldehyde　(TT)　as　an　electron　acceptor.　Under　visible　light　irradiation,　the　donor-acceptor　2D　COF　exhibited　superior　enzymatic　catalytic　activity,　which　could　catalyze　the　oxidation　of　chromogenic　substrates　such　as　3,3′,5,5′-tetramethylbenzidine　(TMB)　by　the　formation　of　superoxide　radicals　and　holes.　Based　on　the　above　property,　the　photoactivated　donor-acceptor　2D　COF　with　enzyme-like　catalytic　properties　was　designed　as　a　robust　colorimetric　probe　for　cheap,　highly　sensitive,　and　rapid　colorimetric　detection　of　glutathione　(GSH);　the　corresponding　linear　range　of　GSH　was　0.4-60　μM,　and　the　limit　of　detection　was　0.225　μM.　This　study　not　only　presents　the　construction　of　COF-based　light-activated　nanozymes　for　environmentally　friendly　colorimetric　detection　of　GSH　but　also　provides　a　smart　strategy　for　improving　nanozyme　activity.　©　2021　American　Chemical　Society.