Reasonable design and construction of artificial enzymes with excellent catalytic activity, good stability and superior biocompatibility is essential for tumor therapy. Single-atom site catalysts (SAs) with well-defined atomic structure and electronic coordination environments have been regarded as a new type of nanozyme that can exhibit excellent catalytic activity like natural enzymes. Here, we constructed a copper-based single-atom site nanozyme (named Cu-N,O/C) that stabilized in hollow carbon nanosphere, and the electronic structure of the single copper active center was precisely controlled by regulating the coordination of nitrogen and oxygen. The well-designed Cu-N₃O active center enables the Cu-N,O/C nanozyme to exhibit multiple extraordinary enzyme-mimicking activities by synchronously catalyzing the generation of reactive oxygen species (ROS), which can effectively inhibit the growth of tumor cell in vitro and in vivo, and the therapeutic effect pronounced enhanced in the weak acidic tumor environment. In addition, the remarkable near infrared spectroscopy (NIR)-photothermal conversion activity of Cu-N,O/C nanozyme significantly enhances the efficiency of cancer cell killing. Therefore, single-atom site nanozyme possesses significant therapeutic efficacy for tumor therapy through multiple ROS and photothermal activity.