Objectives To identify novel lipases with stability and long-chain fatty acids preference by phylogenetic evolution analysis methods from database. Results Thermo-stable Candida antarctica Lipase-A (CALA) was set as a template for gene mining by PSI-BLAST. Based on phylogenetic analysis, three candidate lipases exhibiting 97%, 55%, and 35% identities with CALA, respectively, were selected for overexpression and characterization. Lipase, PhLip from Pseudozyma hubeiensis SY62 showed highest activity towards long-chain fatty acids, and showed maximum activity at pH 9.0 and 60 degrees C, and stability between 40 and 50 degrees C for 4 h and at pH 7-10 for 12 h. Enzymatic hydrolysis of Mucor circinelloides WJ11 oils by PhLip was about twofold higher than that by CALA, with respect to hydrolysis of long-chain fatty acids. Besides, fatty acids with 18 carbons, including oleic acid, linoleic acid, and linolenic acid, were preferred as substrates. Conclusion The current investigation discovered a stable lipase PhLip with long-chain fatty acids preference. PhLip may be a potential candidate for producing polyunsaturated fatty acids from natural oils.