Aspergillus niger lipase (ANL), a widely used hydrolase, was displayed for the first time on the surface of Saccharomyces cerevisiae using a-agglutinin as an anchor protein. Localization of ANL on the cell surface was confirmed by immunofluorescence microscopy. The displayed ANL was confirmed to be active toward tributyrin and p-nitrophenyl caprylate (pNPC). The hydrolytic activity toward pNPC reached 43.8 U/g of dry cell weight after induction by galactose for 72 h. The ANL-displaying cells were characterized for their use as whole-cell biocatalysts. The optimum temperature was 45 °C, and the pH was 7.0. The cells had good thermostability, retaining almost 80% of the full activity after incubation at 60 °C for 1 h, and >80% of the full activity at 50 °C for 6 h. The displayed lipase showed a preference for medium-chain fatty acid p-nitrophenyl esters. Therefore, the produced whole-cell catalyst is likely to have a wide range of applications.