Docosahexaenoic acid (DHA) is an important polyunsaturated fatty acid having pharmaceutical potential. It has gained much scientific attention due to its beneficial use in prevention of many diseases such as dyslexia, Alzheimer, schizophrenia, and dyspraxia. The present article deals with the esterification of DHA-rich fatty acids with glycerol. The reaction has been carried out in biphasic solvent system using immobilized Candida antarctica lipase B (CAL-B) to synthesize glyceride mixture enriched with DHA. The most suitable reaction conditions for esterification of DHA-rich fatty acids with glycerol using immobilized CAL-B were found to be 0.49 g fatty acid mixture with 46mg glycerol, 3mL solvent, 26 mu L buffer, 200mg molecular sieves, 7.0 pH, 50 degrees C temperature, 300 rpm agitation speed, and 50 mg enzyme amount. In 120 h, esterification of 75.4% was obtained with 30% triglyceride, 16.1% diglyceride, and 29.3% monoglyceride in the glyceride mixture. DHA could be concentrated in the glyceride mixture up to 87%.