The development of inorganic antibacterial agents that impart antibacterial properties to biomaterials has attracted wide attention. The paper introduced a kind of hybrid nanosphere antibacterial agent composed of wheat gliadin (WG) and zinc oxide (ZnO), with antibacterial efficacy and low toxicity. The ZnO/WG hybrid nanospheres were environment-friendly integrated by molecular self-assembly co-precipitating and freeze-drying transformation, and were characterized using X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), scanning electron microscope (SEM), atomic absorption spectroscopy (AAS), specific surface and pore size analysis, bacteriostasis test, reactive oxygen species (ROS) determination and safety evaluation. It was found that the prepared hybrid nanospheres were composed of two components, WG and ZnO, with a diameter scope of 100-200 nm; the content of ZnO in the hybrid nanospheres can reach 46.9-70.2% (w/w); the bacteriostasis tests proved that the prepared ZnO/WG nanospheres generating ROS, have a significant inhibitory effect on E. coli and S. aureus; furthermore, the ZnO/WG nanospheres are relatively safe and highly biocompatible in cells and mice. Therefore, the prepared novel ZnO/WG hybrid nanospheres were supposed to apply in the preparation of anti-infective wound dressings, tissue engineering skin scaffold materials, food, and cosmetics preservatives, and so on.