The invention belongs to the technical field of medicines, and discloses an alprostadil lipid nanosphere injection and a preparation method thereof. The alprostadil lipid nanosphere injection is prepared from the following raw materials in parts by weight: 0.002-0.2 part of alprostadil, 40-100 parts of oil for injection, 20-40 parts of emulsifier, 25-50 parts of cosurfactant, 20-30 parts of glycerol, and 780-890 parts of water. The alprostadil lipid nanosphere injection provided by the invention has the particle size smaller than 100 nm, can be sterilized by aseptic filtration, overcomes the instability shortcoming of alprostadil, and improves product stability. Meanwhile, the lipid nanosphere has the particle size smaller than 100 nm, so that the alprostadil lipid nanosphere injection ismore favorable for in vivo non-resveratrol (RES) tissue distribution of alprostadil, reduction of pulmonary circulation inactivation and blood removal, and long-term in vivo circulation of alprostadil. The alprostadil lipid nanosphere injection is suitable for surgical treatment of heart diseases and cranial nerve diseases.