The quantitative measurement of serum hepatitis B surface antigen (HBsAg) is important for monitoring anti-hepatitis B virus (HBV) therapy and evaluating the safety of blood products or blood transfusion. This study is aimed at generating and evaluating a capillary-based chemiluminescence immunoassay for the simple and rapid detection of human serum HBsAg. A simple and rapid capillary chemiluminescence immunoassay (CCIA) was developed using a portable analyzer for quantitatively detecting the levels of HBsAg in human serum. The experimental conditions were optimized, and the sensitivity and specificity of the CCIA were validated in positive HBsAg (HBsAg(+)) and negative HBsAg (HBsAg(-)) human sera. The CCIA quantitatively detected the levels of human serum HBsAg at 0.4-15.0 ng/mL, which resulted in dose-dependent increases in the chemiluminescence (CL) signals, with a sensitivity of 0.3 ng/mL. The assay took only 25 min for the analysis of a single sample. The CCIA only detected HBsAg, but not hepatitis C virus (HCV), human immunodeficiency virus-1 (HIV-1) p24, Treponema (TP) antigens, or other serum proteins and lipids tested with a specificity of 100%. The CCIA for detecting HBsAg was reproducible, with a low intra-assay coefficient of variation (CV) (6.7%) and inter-assay CV (7.4%). The validation of 280 known HBsAg(+) or HBsAg(-) clinical serum samples revealed that the CCIA detected 97.5% of HBsAg(+) sera without false-positives, with a sensitivity and specificity similar to that of the Architect i2000SR HBsAg assay (Kappa value = 0.983). The CCIA is a simple and rapid method for quantitatively measuring the levels of serum HBsAg to monitor anti-HBV therapy and evaluate the safety of blood samples for transfusion.