Ovotransferrin from chicken egg white was separated without using organic solvents. Egg white was diluted with 1 vol. of distilled water (DW), and then homogenized. After removing ovomucin by centrifugation after adjusting the pH to 4.5-5.0, the resulting supernatant was added with ammonium sulfate and citric acid, and then centrifuged after holding overnight at 4 oC. The precipitant, which contains ovotransferrin, was dissolved in DW and the ovotransferrin was re-precipitated using ammonium sulfate and citric acid. The precipitant collected after centrifugation was dissolved with DW, and then desalted and concentrated. The purity of ovotransferrin was determined, the protein identified, and the yield calculated after freeze drying. Over 85% purity and over 83% yield were obtained from the combinations of 5.0% (w/v) ammonium sulfate and 2.5% (w/v) citric acid followed by 2.0% (w/v) ammonium sulfate and 1.5% (w/v) citric acid. The method was simple and cost effective. The isolated ovotransferrin can be used as is or after modifications for various applications such as antimicrobial, anticancer treatments, and iron supplementing agents for human.