In order to realize the selective and accurate detection of H₂O₂ concentration, a fiber-optic evanescent wave biosensor based on horseradish peroxidase (HRP) is developed. First, the ultraviolet resistant quartz fiber with partial core removal is hydroxylated with sodium hydroxide solution and silanized with 3-aminopropyl triethoxysilane, then the fiber is immersed in glutaraldehyde solution for aldehyde cross-linked, and the fiber is moved into HRP solution with selective catalysis for H₂O₂ to fix HRP molecules. Finally, the fiber with HRP is dried at room temperature. The optical fiber biosensor immobilized on HRP can be obtained. The effects of the concentration and fixed time of glutaraldehyde and HRP and the temperature of H₂O₂ solution on the sensitivity of the sensor are studied experimentally. The response time, selection sensitivity, and detection limit of the sensor are tested, and the theoretical model of the sensor is established. The results show that the sensor has a high selective sensitivity to H₂O₂, and the output signal of the sensor has a linear relationship with the concentration of H₂O₂ in the range of 4--20 μmol·L ^-1, the sensitivity is -8.164×10 ^-4 μmol ^-1·L, the relative error is 7.59%, and the detection limit is 4 μmol·L ^-1.