The invention discloses an alprostadil lyophilized microemulsion, and a preparation method and application thereof. The alprostadil lyophilized microemulsion includes: by weight percentage, 0.001% -0.1% of alprostadil, 1.0% -10.0% of grease, 1.0% -20.0% of an emulsifier, 1.0%-20.0% of co-emulsifier, 0.3% -1.6% of a stabilizer, 50.0% -90.0% of a cryoprotectant, and 0.0005 %-0.50 % of a pH conditioning agent. The aforementioned components are prepared into a microemulsion with particle size of 10 nm-50 nm. The microemulsion is filtrated and sterilized, then lyophilized, and ultimately prepared into an injection-used alprostadil lyophilized product with high stability, small re-dissolution particle size (average particle size within 50-100 nm) and good safety. The lyophilized microemulsion is characterized in that: microemulsion drops have a small particle size; the microemulsion can successfully realize filtration and sterilization before lyophilization; the impurity content is low; the side effects are less; the lyophilized product has physical stability and good security and has no hemolysis, irritation, allergy and other toxic irritant reactions; and the storage and transportation is convenient. Before use, adding an appropriate amount of water for injection, glucose solution or physiological saline as need, the lyophilized microemulsion is hydrated, oscillated, diluted, and redissolved into the microemulsion for use of intravenous injection.