The exact mechanism of macrolide-resistant mycoplasma pneumoniae(MRMP) has not been fully determined yet. This study adopted a comprehensive approach of non-targeted proteomics and metabolomics to describe the changes in proteomics and metabolomics in patients with MRMP, patients with MSMP (macrolide sensitive Mycoplasma pneumoniae), and healthy control children.The data were collected from a prospective cohort consisting of 15 children with MRMP, 5 children with MSMP and 10 healthy controls, matched by gender and age. Blood samples were analyzed through a multi-omics platform. The quantification of serum proteins and metabolites was conducted respectively by liquid chromatography-tandem mass spectrometry (LC-MS/MS) based on data independent acquisition and ultra-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). In addition, the proteomics and metabolomics results were analyzed respectively, and a comprehensive analysis was conducted using correlation and pathway analysis. Subsequently, enrichment studies of the Gene Ontology (GO) and Genome Encyclopedia (KEGG) pathways were conducted to clarify the biological correlations of the observation results of this pathway.Compared with the MSMP group and the HC group, 44 significantly different metabolites and 4 significantly different proteins were analyzed in the MRMP group. And the level of Glycocholic acid(GCA) was lowest in MRMP group.The comprehensive analysis of proteomics and metabolomics shows that Cholesterol metabolism and Bile secretion pathwayswere significantly enriched.This comprehensive multi-omics analysis reveals that MRMP exhibits distinct metabolic and proteomic profiles, GCA may be novel biomarker for identifying MRMP. Pathway enrichment analysis highlights cholesterol metabolism and bile secretion as key pathways potentially involved in the drug resistance mechanism. These findings provide new insights into the development of MRMP.