〔Abstract〕 Objective To explore the application effect of multi-level and multi-disciplinary comprehensive treatment in nosocomial infection prevention and control. Methods A total of 1480 inpatients in Jiaozuo People's Hospital from February 2020 to February 2021 were selected as the observation group, and the patients in this group were treated with multi-level and multidisciplinary comprehensive treatment plan to deal with nosocomial infection. A total of 1480 inpatients in Jiaozuo People's Hospital from January 2019 to January 2020 were selected as the control group. The group implementation of routine treatment, prevention and control of nosocomial infection in patients with comparing two groups of patients with nosocomial infection, Ⅰ incision infection, urinary tract infection rate. Result The nosocomial infection, Ⅰ incision infection, urinary tract infection, infection of central venous cannula, infection after use of ventilator, drainage tube infection rate in the observation group were lower than those in the control group, and the differences were statistically significant (P < 0.05); The proportion of hospital and community infection in the observation group were lower than those in the control group, and the differences were statistically significant (P < 0.05). Conclusion The application of multi-level and multi-disciplinary comprehensive treatment in the prevention and control of nosocomial infection can reduce the proportion of nosocomial infection and play a positive role in the timely control of nosocomial infection.