A disposable and sensitive screen-printed carbon electrode for the determination of hydrogen peroxide (H2O2) was fabricated with Prussian blue-modified hydroxyapatite (PB@HAP). PB@HAP was incubated with horseradish peroxidase (HRP). The synergistic effect between HAP and PB facilitated the electron-transfer process and retained the bioactivity of HRP. The proposed biosensor effectively overcame the shortcomings of electron mediator leakage because hydroquinone was added to the base solution. The biosensor showed a low detection limit of 7 μmol/L. A linear calibration plot was obtained over the concentration range, 1–15 mmol/L; the Michaelis constant was 1.31 mmol/L, which indicated that the enzyme retained high bioactivity and excellent appetency toward H2O2. Additionally, the screen-printed biosensor exhibited the characteristics of strong anti-interference, high long-term stability and good reproducibility. Based on the above, PB@HAP has potential for applications in immunosensors and glucose sensors.