This　research　establishes　a　competitive　colorimetric　detection　platform　for　tetracycline　(TC)　residue　analysis　in　milk　and　aqueous　matrices,　with　implications　for　assessing　antibiotic-derived　health　risks　in　dairy　and　potable　water　systems.　According　to　the　developed　colorimetric　competitive　reaction　model,　steady-state　kinetic　analysis　revealed　that　the　Fe-doped　CuO　(Fe-CuO)　nanozymes　exhibit　higher　substrate　affinity　and　faster　reaction　kinetics　than　conventional　enzymes.　The　catalytic　system　utilized　a　bimetallic　nanozyme　composed　of　Fe-CuO,　which　exhibited　enhanced　catalytic　performance,　using　3,3′,5,5′-tetramethylbenzidine　(TMB)　as　the　color-changing　substrate.　Acting　as　the　recognition　component,　TC　competitively　captured　hydroxyl　radicals　(·OH)　produced　during　H2O2　activation　by　the　Fe-CuO　nanozymes,　thus　preventing　the　oxidation　of　TMB　into　its　colored　form　(oxTMB).　The　observed　reaction　pathway　aligned　closely　with　Density　Functional　Theory　(DFT)-predicted　mechanisms,　demonstrating　preferential　oxidation　of　TC　by　·OH.　This　mechanistic　insight　enabled　the　development　of　an　efficient　competitive　sensing　platform.　The　Fe-CuO　nanozyme-based　sensor　showed　a　strong　linear　response　within　the　range　of　1　to　1000　nM　and　achieved　a　remarkable　detection　limit　of　0.274　nM,　indicating　outstanding　analytical　capabilities.　These　results　highlight　its　significant　potential　for　use　in　environmental　monitoring　and　ensuring　food　safety.　©　2025　Elsevier　Ltd