The invention discloses an alprostadil pharmaceutical composition and a preparation method and application thereof, and belongs to the field of medicine and pharmacy. The alprostadil pharmaceutical composition overcomes the defects of complex preparation process and poor stability of a traditional lipid microsphere preparation. The alprostadil pharmaceutical composition is simple in preparation process only needing simple physical mixing without homogenization process. The product can be sterilized through a 0.22 mum millipore filter film, and can spontaneously emulsify during clinical use by dilution with normal saline or glucose solution and other water solutions and slight oscillation, and under the optimal conditions, average particle size is about 0.2 mum, the narrow particle size distribution is narrow, has the prostaglandin E1 (PGE1) encapsulation rate maintained above 95%,, and fully shows lipid microsphere properties. The alprostadil pharmaceutical composition product does not contain water or water solution, and can maintain the stability at room temperature for a long time without PGE1 degradation phenomenon. The alprostadil pharmaceutical composition product can be used in the treatment of diseases such as cerebral vascular disease, severe hepatitis, diabetes complications or erectile dysfunction and the like.