Mono- and diacylglycerol lipase is broadly applied due to its unique substrate specificity. In this study, a putative mono- and diacylglycerol lipase gene prlip was identified from the genome of a food safety strain Penicillium roqueforti. The lipase PrLip was obtained by artificial synthesis strategy and constitutively expressed in Pichia pastoris X33. The lipolytic activity in the broth supernatant reached 22.26U/mL after fermentation for 60h at 30℃. The lipase PrLip was purified by anion exchange chromatography with the purity greater than 90%. The optimal reaction temperature and pH of PrLip were 45℃ and 7.0, respectively, and its thermostable half-life at 40℃ was about 6h. The lipase PrLip was characterized as a mono- and diacylglycerol lipase through hydrolysis and esterification. PrLip had a good tolerance against temperature and resistance to most surfactants, which is promising for potential application in industrial scale.