A novel Cu-doped carbon dots (Cu-CDs) was prepared by simple and eco-friendly one-step hydrothermal method using the natural Astragalus polysaccharides as carbon source and Cu2+ as doping agent. A series of characterization methods (UV-vis, FL, FTIR, TEM and XPS, etc) were used to characterized the optical properties, surface functional groups, morphology and elemental composition of Cu-CDs. The fluorescence quantum yield of Cu-CDs is 22.4% and displayed green fluorescence under 365 nm UV light. L929 cells co-cultured with 200 μg/mL Cu-CDs not only did not show significant cytotoxicity, but also favored cell proliferation. In addition, the hemolysis rate of Cu-CDs for rabbit blood in the concentration range of 0~800 μg/mL is lower than GB/T standard 5%, that is, Cu-CDs has good biocompatibility. Antibacterial results showed that, compared with APS-Cu complexs, Cu-CDs improved the antibacterial activity against Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli), also exhibited better inhibitory activity against Aspergillus flavus (A. flavus) and Aspergillus niger (A. niger). The MIC values of Cu-CDs for the above strains were 60, 100, 125 and 40 μg/mL, respectively. Meanwhile, the antioxidant activity of Cu-CDs was evaluated by the clearance rates of DDPH·, ABTS·+ and OH·. The results showed that the IC50 values of Cu-CDs against DDPH·, ABTS·+ and OH· were 48.6, 6.4 and 3.8 μg/mL, which were much lower than those of pure APS and APS-Cu. In summary, a high biocompatibility, strong antimicrobial and antioxidant activity Cu-CDs based on natural Astragalus polysaccharides and Cu2+ was developed, that offering promising insight into the development of novel antimicrobial and antioxidant agents in biomedical materials and food industry.