The　aim　of　this　study　was　to　develop　a　novel　colorimetric　sensing　method　based　on　enzyme-regulated　instant　generation　of　Turnbull＇s　blue,　serving　as　a　chromogenic　agent,　for　a　sensitive　immunoassay　for　the　determination　of　ochratoxin　A　(OTA).　Unlike　the　traditional　enzyme-linked　immunosorbent　assay　(ELISA),　the　chromogenic　reaction　reported　herein　relies　on　the　immediate　formation　of　Turnbull＇s　blue.　K3[Fe(CN)6]　rapidly　forms　a　coordinate　bond　with　iron(ii),　yielding　a　blue　product.　Meanwhile,　glucose　oxidase　(GOx)　catalyzes　glucose　hydrolysis　to　produce　hydrogen　peroxide　(H2O2),　which　was　used　to　inhibit　the　formation　of　Turnbull＇s　blue　by　oxidizing　iron(ii)　to　iron(iii).　Thus,　Turnbull＇s　blue　was　generated　in　an　enzyme-regulated　manner.　Accordingly,　a　competitive-type　colorimetric　enzyme　immunoassay　was　established　using　a　GOx　based　nanolabel.　Under　optimal　conditions,　the　absorbance　increased　upon　increasing　the　target　OTA　concentration　in　the　range　of　0.01-10　ng　mL-1　with　a　detection　limit　of　8.3　pg　mL-1　estimated　at　the　3Sblank　level.　The　assay　accuracy　was　validated　by　analyzing　spiked　wine　samples.　The　present　results　potentially　provide　novel　insights　into　the　development　of　Turnbull＇s　blue-based　biological　detection　methods　and　colorimetric　immunoassay　strategies.　©　2020　The　Royal　Society　of　Chemistry.