Objective To compare the disinfection effects of four methods on cleaning and disinfecting eye covers. Methods A total of 120 eye covers used by patients were selected and divided into group A, B, C and D, with 30 covers in each group, which were disinfected by using 500 mg/L chlorine disinfectant, 75% ethanol, isopropyl alcohol complex quaternary ammonium salt disinfectant wipes, and moist heat disinfection, respectively. Before disinfection and at 5 min, 4 h and 8 h after disinfection, microbial culture and ATP fluorescence detection were used to compare the qualified rates of disinfection, and the disinfection costs of the four groups were also compared. Results At 5 min after disinfection, the unqualified rates of disinfection of the four groups according to microbial culture and ATP fluorescence detection were 0; at 4 hours after disinfection, the unqualified rate of disinfection in group B was significantly higher than that of group D （P<0.008）;and at 8 hours after disinfection, the unqualified rates of disinfection in group A, B and C weresignificantly higher than that of group D （all P<0.008）. Conclusion The disinfection effect of moist heat disinfection method is significantly better than 500mg/L chlorine disinfectant, 75% ethanol and isopropyl alcohol complex quaternary ammonium salt disinfectant wipes, and it is simple and feasible.