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.