Coronary artery disease (CAD) is a very common cardiovascular disease. Clinically, the content of low-density lipoprotein-cholesterol (LDL-C) is often used as a predictor of CAD. However, severe CAD has been observed in patients with normal level of LDL-C. In this study, the serum lipoproteins stained with Sudan Black B (SBB) were separated and characterized by using asymmetrical flow field-flow fractionation (AF4) coupled with ultraviolet visible (UV/Vis) detector (AF4-UV/Vis). The effects of SBB staining conditions and AF4 operation conditions on AF4 analysis of serum lipoproteins were systematically investigated. The membrane fouling was alleviated by adjusting SBB staining conditions, which enhances the repeatability and accuracy of AF4 analysis of serum lipoproteins. Moreover, the baseline separation of LDL and high-density lipoprotein (HDL) was obtained by AF4 under optimized operation conditions. The results showed that the LDL/HDL value of healthy people was lower than that of CAD patients. The results revealed that the combination of AF4-UV/Vis and SBB staining is a promising approach for the separation and characterization of serum lipoproteins, which is beneficial to screening potential predictors of CAD.