The search for and characterization of new lipases with excellent properties has always been urgent and is of great importance to meet industrial needs. In this study, a new lipase,lipB,fromPseudomonas fluorescensSBW25, belonging to the lipase subfamily I.3, was cloned and expressed inBacillus subtilisWB800N. Enzymatic properties studies of recombinant LipB found that it exhibited the highest activity towardsp-nitrophenyl caprylate at 40 °C and pH 8.0, retaining 73% of its original activity after incubation at 70 °C for 6 h. In addition, Ca2+, Mg2+, and Ba2+strongly enhanced the activity of LipB, while Cu2+, Zn2+, Mn2+, and CTAB showed an inhibiting effect. The LipB also displayed noticeable tolerance to organic solvents, especially acetonitrile, isopropanol, acetone, and DMSO. Moreover, LipB was applied to the enrichment of polyunsaturated fatty acids from fish oil. After hydrolyzing for 24 h, it could increase the contents of polyunsaturated fatty acids from 43.16% to 72.18%, consisting of 5.75% eicosapentaenoic acid, 19.57% docosapentaenoic acid, and 46.86% docosahexaenoic acid, respectively. The properties of LipB render it great potential in industrial applications, especially in health food production.