Realizing the rapid and on-site detection of biothiols in complex biological and food samples using simple assays and devices remains a major challenge. In this study, biothiols containing sulfhydryl groups were found to be able to inhibit the photo-triggered oxidation of 3,3',5,5'-tetramethylbenzidine (TMB). Based on the discovery, using the commercially available and low-cost TMB as the chromogenic substrate, an enzyme-free colorimetric approach was developed for the rapid determination of biothiols. The method does not involve the introduction of any natural enzymes, nanoenzymes, and external oxidants. The mechanisms of the photoinduced oxidation of TMB and the detection of biothiols were proposed. Furthermore, a smartphone-based portable device integrated with test strips was constructed by the 3D printing technique. This device can simultaneously meet the requirements of the photocatalytic oxidation reaction of TMB and the detection of biothiols. The entire process only takes less than 5 min. The successful detection of cysteine in urine and milk samples demonstrates the great potential of the device in the on-site assays.