Separation and concentration of biotechnological products are the most important steps of purification stage in downstream processing. In the present study, three nisin separation and concentration methods including salting out by ammonium sulfate, solvent extraction (at 20 °C, 4 °C, and − 10 °C) and direct chromatography of culture medium were compared with each other. According to our results, nisin precipitation by ammonium sulfate at 40% saturation was the most efficient method (yield = 90.04%, purification fold = 168.80). Low yield and fold purification values were obtained by solvent extraction with chloroform (yield = 24.23%, fold purification = 37.43 at − 10 °C) and direct cation exchange chromatography of culture medium (yield = 20.00%, fold purification = 1.80). Also, performing purification steps in low pH values and acidic conditions (pH = 3.0) is essential for efficient nisin purification.