Objective To investigate the application effect of Methotrexate combined with Compound Polymyxin B Ointment in the treatment of moderate and severe atopic dermatitis in children. Methods A total of 90 children with atopic dermatitis in the Department of Dermatology, Affiliated Hospital of Jiujiang University and the Department of Pediatrics of Jiujiang Maternal and Child Health Care Hospital from December 2018 to December 2020 were selected as the research objects. They were divided into the experimental group (n=45) and the control group (n=45) by computer grouping method. The control group was treated with Methotrexate. The experimental group was treated with Methotrexate combined with Compound Polymyxin B Ointment. The changes of serum related indexes (interleukin-4 [IL-4], interleukin-13 [IL-13]) and eosinophil levels, the improvement of severity scoring of atopic dermatitis (SCORAD) and eczema area and severity index (EASI) score, the occurrence of adverse reactions after medication and score changes of children′s dermatological life quality index (CDLQI) were compared between the two groups before and after treatment.Results The serum-related indicators such as IL-4 and IL-13 and the level of eosinophils in the experimental group after treatment were lower than those in the control group, the differences were statistically significant (P＜0.05). The SCORAD score, EASI score and CDLQI score in the experimental group after treatment were lower than those in the control group, the differences were statistically significant (P＜0.05). There was no significant difference in the incidence of adverse reactions between the two groups (11.11% [5/45] vs. 6.67% [3/45]). Conclusion Methotrexate combined Compound Polymyxin B Ointment in the treatment of moderately severe atopic dermatitis is effective, which can effectively control the patient′s serum inflammatory biomarkers and eosinophil level, and significantly relieve the clinical symptoms and signs. At the same time, the quality of life of children can be significantly improved, and will not bring serious adverse reactions to children. The effectiveness and safety are high. It is suggested that it can be popularized and applied in clinic as appropriate.