A simple, colorimetric method is developed for the determination of deoxyribonuclease I (DNase I) activity based on the novel finding that DNase I can promote the photoinduced synthesis of gold nanoparticles (AuNPs). In the absence of DNase I, a phosphorothioate (PS) DNA probe remains intact and captures Au(iii) through a strong Au-thiol interaction, which prevents the photoinduced synthesis of AuNPs, leaving the sample in a colorless state. On the other hand, in the presence of DNase I, the PS DNA probe is cleaved into small fragments that are removed via a simple purification process. The resulting solution, after the incubation with HAuCl4 and threonine (Thr), forms AuNPs by UV light irradiation with the aid of Thr which acts as a catalyst for the Au(iii) reduction process. As a result, a red-colored suspension is produced. By monitoring the color changes of the samples with the naked eye, the DNase I activity was conveniently determined. In addition, the clinical utility of this simple, yet highly efficient colorimetric strategy was verified by reliably quantifying the DNase I activities in a bovine urine sample. Importantly, the working principle designed for the determination of DNase I activity was successfully expanded for the detection of target nucleic acids, ensuring the universal applicability of the developed assay system.