Ovotransferrin is one of the major egg white proteins that have antimicrobial activity as well as iron binding capability. The objective of this study was to develop a simple and easy method to separate ovotransferrin without using organic solvents. Egg white was separated from yolk, added in a 1:1 ratio to distilled water (DW), and then homogenized. The ovomucin in the diluted egg white was removed by centrifugation, adjusting the pH to 4.5 to 5.0. The resulting supernatant was added to different ratios of ammonium sulfate and citric acid, and then centrifuged after holding overnight at 4°C. The precipitant, which contains ovotransferrin, was dissolved in DW, and ovotransferrin was precipitated using different ratios of ammonium sulfate and citric acid. The precipitant collected after centrifugation was dissolved with DW and subjected to ultrafiltration to remove salts and concentrate the solution. The purity of the ovotransferrin was determined using SDS-PAGE, the protein identified using Western blot, and the estimated yield calculated by weighing the ovotransferrin after freeze drying. Over 85% purity and over 83% yield were obtained from the combinations of 5.0% (wt/vol) ammonium sulfate and 2.5% (wt/vol) citric acid followed by 2.0% (wt/vol) ammonium sulfate and 1.5% (wt/vol) citric acid. Activity of the ovotransferrin showed similar activity with previously separated ovotransferrin. However, this method is simpler and more cost effective than the previous method. The isolated ovotransferrin can be used as is or after modifications for various applications such as antimicrobial treatments, anticancer treatments, and iron-supplementing agents for humans.