Abstract Yeast whole cell biocatalysts, which intracellularly overproduced a recombinant lipase with a pro-sequence from Rhizopus oryzae IFO4697 (rProROL) were constructed, and the content of active lipase in Saccharomyces cerevisiae cells was maximized by optimizing the cultivation procedure. rProROL was overproduced intracellularly under the control of the 5′-upstream region of the isocitrate lyase gene of Candida tropicalis ( UPR-ICL ) as the inducible system and the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter as the constitutive expression system. Enhancement of expression level of ProROL gene at the initial cultivation phase inhibited rProROL accumulation in yeast cells both in GAPDH promoter system by using high glucose concentration at 30 °C and in UPR-ICL system by using non-fermentable carbon sources. The highest intracellular lipase activity of 350.6 IU/l was obtained in the inducible UPR-ICL system with an initial glucose concentration of 0.5% at 30 °C. To prepare the efficient whole cell biocatalyst by intracellular overproduction of lipase, utilization of inducible UPR-ICL and the optimization of cultivation conditions such as temperature, carbon source and its initial concentration are important.