It is highly desirable but still remains challenging to tune nanozymes with superior catalytic activity via simple operation. Herein, the oxidase-like activity of Ru nanoparticles supported on porous carbon (Ru/PC) was significantly modulated via a facile partial oxidation process. Due to the small size, uniform dispersion, high electron density on the surface, as well as abundant interfacial heterostructure between Ru and RuO 2 , the partially oxidized Ru/PC (P−Ru/PC) showed excellent oxidase-mimicking activity in triggering 3,3′,5,5′-tetramethylbenzidine (TMB) oxidation, with a low K m of 0.138 mM and a high V max of 2.82 × 10 −7 M• s −1 . Theoretical calculations reveal that both the absorption and subsequent activation of molecular oxygen are more favorable on the interfacial heterostructure of P−Ru/PC than on the Ru/PC surface. Moreover, the oxidase-like activity of P−Ru/PC was found to be suppressed by melatonin, along with a fading color of oxidized TMB (TMB ox ). As a result, a novel colorimetric system consisting of P−Ru/PC and TMB was established for melatonin detection for the first time, which shows a wide linear range of 10−220 μM and a low detection limit of 0.023 μM. This work not only provides a simple but smart strategy of interface engineering to tune the oxidase-like activity of Ru/PC, but also expands the application of Ru oxidase mimics in pharmaceutical analysis.