An organic solvent-stable lipase from Proteus vulgaris K80 was used to produce the omega-3 polyunsaturated fatty acid ethyl esters (omega-3 PUFA EEs). First, the lyophilized recombinant lipase K80 (LyoK80) was used to perform the transesterification reaction of menhaden oil and ethanol. LyoK80 produced the omega-3 PUFA EEs with a conversion yield of 82 % in the presence of 20 % water content via a three-step ethanol-feeding process; however, in a non-aqueous condition, LyoK80 produced only a slight amount of the omega-3 PUFA EEs. To enhance its reaction properties, the lipase K80 was immobilized on a hydrophobic bead to derive ImmK80; the biochemical properties and substrate specificity of ImmK80 are similar to those of LyoK80. ImmK80 was then used to produce omega-3 PUFA EEs in accordance with the same transesterification reaction. Unlike LyoK80, ImmK80 achieved a high omega-3 PUFA EE conversion yield of 86 % under a non-aqueous system via a one-step ethanol-feeding reaction. The omega-3 PUFA EEs were purified up to 92 % using a urea complexation method.