The invention relates to a disinfection method of surgical instruments. The surgical instruments are counted and classified, and different types of instruments are separately disinfected. The disinfection method comprises the following steps: placing one surgical instrument to be disinfected into a closed spraying box, cleaning by spraying for 15 to 25 min by using a biological enzyme cleaning agent, and thoroughly discharging the biological enzyme cleaning agent; cleaning by spraying for 5 to 10 min by using medical iodophor, and thoroughly discharging the iodophor; placing the surgical instrument to be disinfected into ozone oxidized water, soaking for 5 to 10 min, and then taking out the surgical instrument; drying the surgical instrument, and oiling the surgical instrument; performing irradiation treatment on the surgical instrument to be disinfected for 2 to 5 min by using a high-energy electron irradiation accelerator; and finally orderly placing the instrument into a surgical instrument box or a dedicated packaging bag and sealing according to the type of the instrument. The disinfection method of the surgical instruments has the advantages of simple, safe and reliable operation, low operation and implementation cost, great convenience in use, short working time, capability of cleaning and disinfecting various medical instruments, suitability for being popularized in the medical field, and capability of preventing the cross infection caused by the un-thorough disinfection of the surgical instruments.