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Gubenfangxiao decoction (GBFXD) is a traditional Chinese medicine formula derived from Yupingfengsan, an ancient formula widely used to treat respiratory diseases. In recent years, GBFXD has been applied to efficaciously and safely treat asthma. However, the mechanism of GBFXD is still not fully elucidated. The aim of this study was to employ the label-free proteomic method to explore the protective mechanism of GBFXD in respiratory syncytial virus (RSV)-ovalbumin (OVA) induced chronic persistent asthmatic mice. After RSV-OVA challenge, mice were orally administered GBFXD at a dose of 36 g/kg accompanied with OVA nasal spray once every 3 days for 28 days. The label-free proteomics-based liquid chromatography-tandem mass spectrometry method was used to explore the differentially abundant proteins (DAPs) in the serum from model mice compared with that in control mice (M:C), and in GBFXD-treated mice compared with that in model mice (G:M). The mass spectrometry proteomics data have been deposited to the ProteomeXchange with identifier PXD013244. A total of 69 significant DAPs were identified including 39 in M:C, 46 in G:M, and 16 common differential proteins. Bioinformatics analysis revealed that the DAPs of M:C were mainly involved in inflammatory response and were related to lipid metabolism. However, the DAPs of G:M mostly participated in stress response, inflammatory response, and epithelial cell proliferation. Serum levels of Apoa-1, Apoc-1, Cfd, and Lrg1, EGFR and Lrg1 in the lungs were consistent with the results of proteomic analysis. Apoa-1 and Apoc-1 were closely related to cholesterol transport, lipid metabolism balance, and airway epithelial integrity; Cfd participated in immune response, affecting the occurrence and development of inflammation; EGFR and Lrg1 were involved in epithelial cell proliferation, influencing the process of airway remodeling. In summary, these results indicated that GBFXD may affect inflammatory and immune response of asthma by regulating cholesterol transport and complement factor activation. Furthermore, it could repair damaged airway epithelium and avoid airway remodeling to prevent and treat asthma.