An extracellular lipase derived from Bacillus circulans, isolated from marine macroalga, Turbinaraia conoides, was used to prepare n-3 polyunsaturated fatty acid (PUFA) concentrates from sardine oil triglycerides. The enzyme was purified 132-fold with specific activity of 386LU/mg. The purified lipase was able to enrich sardine oil with 37.7±1.98% 20:5n-3 and 5.11±0.14% 18:3n-3 in the triglyceride fraction after 3h of hydrolysis. Lower hydrophobic constants of n-3 fatty acids (18:3n-3 log P =5.65; 20:5n-3 log P =5.85, respectively) than n-6 (20:4n-6 log P =6.16) resulted in higher hydrolytic resistance of the former toward lipase, leading to their enrichment in the triglyceride fraction. Lipase-catalysed hydrolysis of sardine oil for 3h, followed by urea complexation, provided free fatty acids containing 51.3±4.65% 20:5n-3. The purified methyl ester of 20:5n-3 (68.29±2.15%) from the urea concentrate was attained by chromatography on argentated neutral alumina.