A novel lipase from Bacillus licheniformis NCU CS-5 was expressed in different Escherichia coli cells. The recombinant enzyme achieved a high activity (161.74 U/mL) with protein concentration of 0.27?mg/mL under optimal conditions at the large-scale expression of 12?h. The recombinant lipase showed optimal activity at 40 ℃ and pH 10.0, and maintained more than 80% relative activity after 96?h of incubation at pH 9.0-10.0. This typical alkaline lipase was activated under medium temperature conditions (30 and 45 ℃ for 96?h). The lipase exhibited a degree of adaptability in various organic solvents and metal ions, and showed high specificity towards triglycerides with short and medium chain fatty acids. Among different substrates, the lipase showed the strongest binding affinity towards pNPP (Km?=?0.674?mM, Vmax?=?950.196?μM/min). In the experiments of its application in enhancing fatty acids flavor release for low-fat cheeses, the lipase was found to hydrolyze cheeses and mainly increase the contents of butyric acid, hexanoic acid, caprylic acid and decanoic acid. The results from NMR and GC provided the possibility of enhancing fatty acids flavor released from low-fat cheeses by the lipolysis method.