Geotrichum sp. lipase (GSL) was first displayed on the cell wall of Pichia pastoris on the basis of the a-agglutinin anchor system developed in Saccharomyces cerevisiae . Surface display levels were monitored using Western blotting, immunofluorescence miscroscopy, and fluorescence-activated cell sorting analysis. Lipase activity of the yeast whole cells reached a maximum at 273 ± 2.4 U/g of dry cells toward olive oil after 96 h of culture at 30 °C, with optimal pH and temperature at 7.5 and 45 °C, respectively. Displayed GSL exhibited relatively high stability between pH 6.0 and 8.0 and retained >70% of the maximum activity. The surface-displayed lipase retained 80% of its original activity after incubation at 45 °C for 4 h. Moreover, the GSL-displaying yeast whole cells were then used as a biocatalyst to enrich eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) from fish oil on the basis of selective hydrolysis. As a result, EPA and DHA increased from 1.53 and 24.1% in the original fish oil to 1.85 and 30.86%, which were increases of 1.21- and 1.29-fold, respectively. The total yield of EPA and DHA reached 46.62%.