Triacylglycerols are considered one of the most promising feedstocks for biofuels. Phospholipid:diacylglycerol acyltransferase (PDAT), responsible for the last step of triacylglycerol synthesis in the acyl-CoA-independent pathway, has attracted much attention by catalyzing membrane lipid transformation. However, due to lack of biochemical and enzymatic studies, PDAT has not carried forward in biocatalyst application. Here, the PDAT from Saccharomyces cerevisiae was expressed in Pichia pastoris. The purified enzymes were studied using different acyl donors and acceptors by thin layer chromatography and gas chromatography. In addition of the preferred acyl donor of PE and PC, the results identified that ScPDAT was capable of using broad acyl donors such as PA, PS, PG, MGDG, DGDG, and acyl-CoA, and ScPDAT was more likely to use unsaturated acyl donors comparing 18:0/18:1 to 18:0/18:0 phospholipids. With regard to acyl acceptors, ScPDAT preferred 1,2 to 1,3-diacylglycerol (DAG), while 12:0/12:0 DAG was identified as the optimal acyl acceptor, followed by 18:1/18:1 and 18:1/16:0 DAG. Additionally, ScPDAT reveals esterification activity that can utilize methanol as acyl acceptor to generate fatty acid methyl esters. The results fully expand the enzymatic selectivity of ScPDAT and provide fundamental knowledge for synthesis of triacylglycerol-derived biofuels.