Human serum albumin (HSA), the most abundant protein in human serum, accounting for about 40%~60% of the total serum protein content. When purified by commercialized albumin affinity medium (Cibacron Blue F3GA), the yield of albumin is low, meanwhile, the ligand of Cibacron Blue F3GA had disadvantages of high toxicity and easily to fall off. In this study, a novel electrostatic coupling affinity medium was prepared and used to purify albumin from human serum by one-step chromatography. New electrostatic coupling affinity medium DASA-Sepharose (3,5-diaminobenzoic acid n-octyl succinic anhydride-Sepharose) was prepared with n-octyl succinic anhydride as affinity ligand coupled to agarose microsphere with 3,5-diaminobenzoic acid as spacer arm. The carboxyl functional group on the DASA-Sepharose spacer arm adsorbed albumin through electrostatic interaction, and then cooperated with the n-octyl succinic anhydride affinity ligand to achieve electrostatic coupling affinity adsorption, which greatly improved the adsorption capacity and maintained the high specificity of affinity adsorption. The effects of different NaCl concentrations and pH values on adsorption equilibrium were investigated with BSA as model protein. When NaCl concentration was 0.025~0.06 mol/L, the saturated adsorption capacity (Qm) was almost unaffected. When NaCl concentration was 0.1 mol/L or above, Qm decreased significantly. The Qm for BSA reached 75.43 mg/mL medium in 20 mmol/L PBS at pH=5.00. Compared with the Qm (20 mg/mL medium) of the commercialized albumin affinity medium Cibacron Blue F3GA, the Qm of DASA-Sepharose increased by about 2.7 times. The HSA could be directly extracted from human serum using the novel electrostatically coupled affinity chromatography medium with high purity (98.20%) and high yield (94.34%).The secondary structure and small molecule drug binding activity of purified albumin were determined by circular dichroism spectrum and warfarin sodium method, which were basically consistent with the standard human serum albumin. The results demonstrated that the electrostatic coupling affinity chromatography can be efficiently used for HSA purification from human serum, which provided a new approach for the HSA separation from plasma.