Chronic hepatitis B is a significant public health problem and complex pathologic process, and unraveling the underlying mechanisms and pathophysiology is of great significance. Data independent acquisition mass spectrometry (DIA-MS) is a label-free quantitative proteomics method that has been successfully applied to the study of a wide range of diseases. The aim of this study was to apply DIA-MS for proteomic analysis of patients with chronic hepatitis B. We performed comprehensive proteomics analysis of protein expression in serum samples from HBV patients and healthy controls by using DIA-MS. Gene Ontology (GO) terms, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, and protein network analysis were performed on differentially expressed proteins and were further combined with literature analysis. We successfully identified a total of 3786 serum proteins with a high quantitative performance from serum samples in this study. We identified 310 differentially expressed proteins (DEPs) (fold change > 1.5 andPvalue < 0.05 as the criteria for a significant difference) between HBV and healthy samples. A total of 242 upregulated proteins and 68 downregulated proteins were among the DEPs. Some protein expression levels were significantly elevated or decreased in patients with chronic hepatitis B, indicating a relation to chronic liver disease, which should be further investigated.