Myrosinase (Myr) is a type of critical β-thioglucosidase enzyme activator essential for sustaining many functional foods to perform their health-promoting functions. An accurate and reliable Myr test is meaningful for food quality and dietary nutrition assessments, whereas the currently reported methods do not guarantee specificity and have high reliance on instrumentation, which are not suitable for rapid and onsite Myr screening especially in complex systems from various sources. Herein, we present a unique NIR-II absorption-based photothermal-responsive colorimetric biosensor for anti-interference onsite Myr determination and realization of rapid visualized outputs with the aid of smartphone calculation. Typically, assisted by glucose oxidase (GOx), Myr specifically converts the sinigrin substrate into hydrogen peroxide (H2O2) that can oxidize 3,3',5,5'-tetramethylbenzidine (TMB) catalyzed by AuNPs to form a charge transfer complex (CTC) with NIR-II absorption and photothermal characters. Delightfully, such a proposed method is able to determine Myr within a wide range of 0 to 172.5 mU/mL with a detection limit down to 2.96 mU/mL. Moreover, simple, rapid, and real-time visual Myr identification in actual food-sourced samples could also be readily achieved by smartphone readout processing, with the promising advantages of anti-interference, high accuracy, and low cost as well as labor-saving and intelligence engagement, thus providing great feasibility for precise measurement in complex and dynamic dietary sample analysis. Overall, our proposed method presents a novel technology for onsite dietary Myr enzyme profiling, which is promising to be applied in the food industry for nutritional composition profiles, freshness evaluation, and quality assessment.