The invention belongs to the field of medicinal preparation, and specifically relates to a polyene phosphatidyl choline injection, and a preparation method thereof. The polyene phosphatidyl choline injection comprises polyene phosphatidyl choline, a solubilizer, an antiseptic, and a stabilizing agent; the solubilizer is selected from deoxycholic acid, deoxysodium cholate, dehydrocholic acid, sodium dehydrocholate, cholic acid, or sodium cholate; and the stabilizing agent is selected from amino acid, or a sodium salt thereof, or trometamol. Compared with a control group without the stabilizing agent, stability of the polyene phosphatidyl choline injection after high temperature sterilization and long-term storage is increased significantly, formula and preparation method are both simple, and the polyene phosphatidyl choline injection is convenient for industrialized production.