Objective: This study aims to explore the effect of Prussian blue nanozyme on osteogenic differentiation of MC3T3-E1 cells in an oxidative microenvironment. Methods: Biocompatibility of different concentrations of Prussian blue nanozymes was detected by CCK-8 assay and flow cytometry; mimetic enzyme activity of Prussian blue nanozymes at different concentrations was measured by xylenol orange assay and WST-8 assay, and intracellular reactive oxygen species scavenging activity of Prussian blue nanozymes was detected by DCFH-DA probe immunofluorescence assay; and the osteogenesis differentiation of MC3T3-E1 cells under oxidative microenvironment was evaluated by alkaline phosphatase (ALP) staining and activity assay, alizarin red staining and real-time fluorescence quantitative reverse transcription PCR. Results: CCK-8 and flow cytometry showed good biocompatibility of Prussian blue nanozyme between 0-100 μg/mL concentrations; xylenol orange ssay and WST-8 assay showed good mimetic enzymatic activity of Prussian blue nanozyme in a concentration and time-dependent manner; DCFH-DA probe immunofluorescence showed that Prussian blue nanozyme also had good ability to scavenge intracellular reactive oxygen species; ALP staining and activity assay, alizarin red staining and real-time fluorescence quantitative reverse transcription PCR showed that Prussian blue nanozyme can significantly enhance osteogenesis differentiation of MC3T3-E1 cells in oxidative microenvironment. Conclusion: Prussian blue nanozyme can enhance the antioxidant activity of MC3T3-E1 cells and promotes the osteogenic differentiation of cells in an oxidative environment.