Phosphatidylserine (PS) has significant biological and nutritional effects and finds wide applications in the food, pharmaceutical, and chemical industries. To produce high-value PS efficiently, phospholipase D (PLD)-induced transphosphatidylation of low-value phosphatidylcholine (PC) with L-serine has been explored. In this research, we purified recombinant PLD from Streptomyces antibioticus SK-3 using ion exchange chromatography and gel filtration chromatography. Subsequently, we thoroughly characterized the purified enzyme and optimized the transphosphatidylation conditions to identify the most favorable settings for synthesizing PS in a biphasic system. The purified recombinant PLD displayed a robust transphosphatidylation function, facilitating efficient catalysis in the synthesis of PS. Under the optimal conditions (butyl acetate/enzyme solution 1:1, L-serine 160 mg/mL, soybean lecithin 2 mg/mL, and MgCl2 15 mM, at 50 °C for 2.5 h with shaking), we achieved a conversion rate of 91.35% and a productivity of 0.73 g/L/h. These results demonstrate the applicability of the process optimization strategy for using the candidate enzyme in the efficient synthesis of PS. Overall, this study presents a novel and scalable approach for the efficient large-scale synthesis of PS.