A method that could separate the subclasses of high density lipoprotein (HDL) rapidly by microfluicic chip electrophoresis was reported. Combined with laser induced fluorescence detection system, using the microfluicic chip designed independent, using 40 mmol/L Tricine, 50 mmol/L MEG and 0.2 mmol/L SDS (pH 8.5) as sample buffer solutions, using 40 mmol/L Tricine, 50 mmol/L MEG and 0.01 mmol/L SDS (pH 8.5) as separation buffer solutions, the two subclasses, HDL₃ and HDL₂ could be separated in 4 min. HDL subclasses could be separated with high efficiency and good reproducibility. The operation was easy and cost little. This method could meet the demand of the clinical examination of HDL subclasses, with good clinical value.