Fraction V supernatant is an effluent of Cohn fractionation in plasma protein industry. Due to its high ethanol concentration, further recovery of the residual protein has been regarded as non-economical. In this work, a recovery of human serum albumin (HSA) from fraction V supernatant by ion exchange chromatography was reported, which had not been reported in the literature to our knowledge. Firstly, bovine serum albumin (BSA) was used as model protein to compare the adsorption capacity of three different types of chromatographic media in different ethanol-aqueous solutions. The adsorption capacity of the hydrophobic medium to BSA in ethanol-aqueous solution was very weak, and the increase of ethanol concentration led to the adsorption capacity approaching to 0. The cation exchange medium had a high adsorption capacity at low ethanol concentration, but decreased quickly with the increase of the ethanol concentration. In contrast, the anion exchange medium showed the best adsorption performance, and the adsorption capacity in 40% ethanol-aqueous solution still reached 34.66 mg/mL. Further experiments showed that the adsorption of BSA on the anion exchange medium in the presence of ethanol could be described by Langmuir isothermal adsorption equation. The anion exchange medium DEAE Sepharose Fast Flow was packed into a chromatographic column. Real purification of Cohn fraction V supernatant was performed. The Cohn fraction V supernatant, containing about 40% ethanol, was directly loaded to the anion exchange column. A two-step elution strategy was used. The first elution was pH change from 7.0 to 4.5 to obtain the target product human serum albumin, and the second elution was to increase the concentration of sodium chloride from 0 to 1 mol/L to elute glycoproteins. The purity of HSA was 96.35% by electrophoresis, and the activity of binding to the ligand warfarin was comparable to that of commercial HSA product by Cohn fractionation. The total recovery was 43 mg/L Cohn fraction V supernatant.