Production of omega-3 polyunsaturated fatty acids concentrates from oil of Schizochytrium limacinum was optimized to obtain the highest omega-3 polyunsaturated fatty acid (omega-3PUFA) enrichment ratio. In this process, the content of docosapentenoic acid (DPA), the content of docosahexenoic acid (DHA) in the final product, the percentage recovery of the heavy phase, and the omega-3PUFA enrichment ratio were optimized. Single-factor experiments were carried out to study the effects of evaporator temperature, feeding rate, feeding temperature, and roller speed. A central composite design was used to optimize technological conditions. Under optimum conditions, the highest omega-3PUFA enrichment ratio (92.98%) was obtained at an evaporator temperature of 110.4 degrees C, a feeding rate of 78.7 mL/h, a roller speed of 350 rpm, a feeding temperature of 80 degrees C, and an operating pressure 10 Pa.