The invention discloses a preparation method of phosphatidylserine. The preparation method comprises the following steps: 1) mixing fish muscle tissues, hexane and isopropanol, carrying out extraction, filtering, and taking a supernatant M1; 2) mixing the supernatant M1 obtained in the step 1) with sodium salt solution, then standing, and taking an upper layer, so that a crude product M2 is obtained; and 3) enabling the crude product M2 obtained in the step 2) to sequentially pass through ion exchange resin and a silica gel column, so that the phosphatidylserine is obtained. According to the invention, the fish muscle tissues, hexane and isopropanol are mixed, extracted and filtered, the supernatant M1 is taken, then the supernatant M1 and the sodium salt solution are mixed and then standing is carried out, then the supernatant after standing is further extracted, so that the crude product M2 containing the phosphatidylserine is obtained and further the crude product passes through the ion exchange resin and the silica gel column, so that the phosphatidylserine with higher purity is obtained; meanwhile, not only is an operation method simple, but also raw materials are easily available, and production efficiency is greatly improved.