Objective: To comparatively analyze the application value of enzyme-linked immunosorbent assay and chemiluminescence method in hepatitis B detection. Method: 120 patients carrying hepatitis B virus who were admitted to our hospital from January 2022 to December 2023 but did not receive antiviral treatment were selected as the research subjects. All the research subjects used enzyme-linked immunosorbent assay and chemiluminescence method respectively to detect Hepatitis B virus markers in patients, including Hepatitis B surface Antigen (HBsAg) and hepatitis B surface Antibody ( HBsAb), Hepatitis Be Antigen (HBeAg), Hepatitis Be Antibody (HBeAb), and hepatitis B core Antibody (HBcAb). The positive detection rates of hepatitis B virus markers and the minimum detectable concentrations of serum HBsAg by enzyme-linked immunosorbent assay and chemiluminescence were analyzed and compared, and the consistency of the detection results of the two methods was also compared. Result: Compared with the enzyme-linked immunosorbent assay, the chemiluminescence method significantly increased the positive detection rates of serum HBsAg, HBeAg and HBcAb in patients (P < 0.05). The detection results of serum HBsAg, HBsAb, HBeAg, HBeAb and HBcAb by enzyme-linked immunosorbent assay and chemiluminescence method have good consistency. The minimum detectable concentrations of serum HBsAg by enzyme-linked immunosorbent assay (ELISA) and chemiluminescence were 0.10IU·mL-1 and 0.03IU·mL-1 respectively. The chemiluminescence method was more sensitive in detecting serum HBsAg. Conclusion: The detection results of hepatitis B virus markers by enzyme-linked immunosorbent assay (ELISA) and chemiluminescence method have good consistency. Moreover, compared with ELISA, chemiluminescence method has a higher positive detection rate of HBsAg, HBeAg and HBcAb, and is more sensitive to the detection of serum HBsAg.