Phospholipase D (PLD) derived from Streptomyces has great application potential in food, health products, medicine and other industries, and the commonly used heterologous expression host is E. coli. However, there are problems such as long fermentation time, cumbersome separation and purification steps, and high cultivation cost. Therefore, the effects of intracellular expression, secretory expression and surface display of E. coli on PLD enzyme activity and phosphatidylserine (PS) catalytic synthesis efficiency were comparatively studied. A single factor method was used to derive the optimal induction conditions for PLD expression modes, specifically, the bacterial concentration OD₆₀₀ was 0.7, IPTG concentration was 0.3 mmol·L^-1 and the temperature was 20 ℃. The fermentation time curve under optimal conditions showed that the PLD enzyme activity by the above three methods reached the highest value when the expression was induced for 8 h, which were 0.35, 0.23, and 0.11 U·mL^-1, respectively. Scanning electron microscopy showed that the structure of E. coli cells expressing PLD has been damaged to a certain extent. Among them, the deformation and collapse of the cell structure of the secretory expression strain is the most serious. In the water-ethyl acetate biphase catalyzed phosphatidylcholine (Phosphatidylcholine, PC) system, the PLD surface display strain in the form of a whole cell obtained the highest PS molar conversion rate of 59.1%, compared with the intracellular expression strain and secretion expression strain in the form of sonicated crude enzyme solution, showing that the expression of the E. coli surface display system PLD has great potential for whole-cell catalytic synthesis of PS with reducing production costs.