Chronic hepatitis B (CHB) remains a major public health problem worldwide. Combined use of nucleoside analogues (NAs) and polyethylene glycolated interferon (PEG-IFNα) can rapidly reduce hepatitis B surface antigen (HBsAg) levels and lead to clinical cure in some advantaged populations. However, the specific molecular mechanisms by which NAs in combination with interferon therapy achieve rapid HBsAg reduction are unknown. To investigate the biomolecular differences associated with combination therapy, we analysed serum protein mass spectra from 25 patients with chronic hepatitis B who experienced a rapid reduction in HBsAg levels after combination therapy. Analyses were performed using data-independent acquisition (DIA)-based liquid chromatography-mass spectrometry before treatment, at week 12 of treatment, and at week 24 of treatment. A total of 3174 proteins were identified in our study. Among these proteins, 54 and 154 dysregulated proteins were detected at week 12 and 24 of treatment, respectively, compared with the pretreatment period.