Aim An immunodetection method was developed to determine the contents of serum HDL subclasses. Methods High density lipoprotein (HDL) subclasses in human serum were separated by two-dimensional agarose-gradient polyacrylamide gel electrophoresis, the molecular size and mass of the HDL subclasses were determined by immunoblotting test and density scanning. Results The variation coefficients of the contents of the HDL subclasses are 4.9%～11.1%. The contents of HDL 2b , HDL 2a , HDL 3a , HDL 3b , HDL 3c in normalipidic serum are 30.80%±6.95%, 21.60%±3.88%, 20.28%±3.69%, 13.54%±2.03%, 7.05%±1.75%, respectively; and that of pre β1-HDL and pre β₂-HDL are 6.67%±1.22%, 4.52%±1.12%, respectively. Conclusion The results suggested the method developed could be used to determine the contents of α-HDL and pre-β HDL subclasses in serum sample.