Colorimetric　immunoassays　are　widely　used　for　biomarker　detection,　offering　advantages　of　simplicity,　sensitivity,　and　cost-effectiveness.　Recent　advancements　focus　on　improving　the　catalytic　activity　of　nanozymes　for　enhancing　the　sensitivity　and　accuracy　of　such　assays.　Bimetallic　CuCo　Prussian　blue　analog　(CuCo　PBA)　has　emerged　as　promising　candidates　due　to　their　excellent　peroxidase-like　activity.　However,　their　instant　synthesis　and　integration　into　immunoassays　for　the　rapid　detection　of　biomarkers　like　carcinoembryonic　antigen　(CEA)　remain　underexplored.　This　study　presents　an　innovative　approach　using　CuCo　PBA　nanozymes　for　colorimetric　immunoassays　with　immediate　generation　and　application.　In　this　study,　CuCo　PBA　nanozymes　were　synthesized　instantly　by　reacting　Cu2+　with　K3[Co(CN)6]　(2O2　and　3,3′,5,5′-tetramethylbenzidine　(TMB),　enabling　sensitive　detection.　The　assay　was　optimized　for　various　parameters,　including　pH,　temperature,　and　material　ratio.　A　linear　response　was　obtained　for　CEA　detection　over　a　concentration　range　of　0.05–60　ng/mL　with　a　limit　of　detection　(LOD)　of　22　pg/mL.　The　integration　of　glucose　oxidase　(GOx)　mediated　the　generation　of　H2O2,　triggering　the　colorimetric　reaction.　This　instantaneous　CuCo　PBA-based　system　effectively　detected　CEA　in　human　serum　samples,　highlighting　its　potential　for　rapid　diagnostic　applications.　This　work　introduces　a　novel　approach　for　rapid　and　sensitive　colorimetric　immunoassays　using　CuCo　PBA　nanozymes　that　are　synthesized　on-demand　and　immediately　applied.　The　system　allows　for　efficient　CEA　detection　with　an　exceptionally　low　detection　limit,　offering　great　potential　for　clinical　diagnostics.　The　instant　generation　and　application　of　CuCo　PBA　nanozymes　in　immunoassays　represent　a　significant　advancement　in　point-of-care　testing　technologies.　©　2025　Elsevier　B.V.