The magnetic Fe3O4 was used as the carrier, and the magnetic cross-linked scPLD aggregates (MCLEA) were prepared by adsorption-aggregation-crosslinking. The enzyme activity recovery of the MCLEA was 72.89%, and the enzyme activity was 437±6.60 U/g. Compared to free scPLD, the MCLEA showed better tolerance at different temperatures and pH levels. However, due to the reduced affinity of the MCLLEA with the substrate, it requires higher concentrations of phosphatidylcholine (PC) and Ca2+ as the reaction substrate to bind to MCLEA. Furthermore, the organic solvent tolerance of the MCLEA was also investigated, and organic solvents such as ethanol, tetrahydrofuran, tert-butanol, ethyl acetate, ether, and toluene were found to improve the enzymatic reactions of MCLEA. These findings provide a reference for the selection of organic solvents in the construction of a biphasic system in the production of phosphatidylserine (PS) catalyzed by scPLD. After 13 cycles of continuous reaction, the activity of the MCLEA remained above 76%, giving a half-life of 331.67 min, which indicated that the immobilizing method developed in this work could improve the stability of the PLD and its reusability in the catalytic reaction.