The invention relates to a method for preparing sterile tacrolimus nano suspension eye drops. The method for preparing the sterile tacrolimus nano suspension eye drops comprises the following main steps: preparing tacrolimus nano suspension by adopting a phase transfer and high pressure homogenization method; removing an organic solvent in the tacrolimus nano suspension by adopting a drying/curing technology, and stabilizing tacrolimus nano particles; sterilizing to obtain sterile tacrolimus nano particles; and finally carrying out sterile redissolution and prepackaging operation, so that the finished product is obtained. By adopting the method for preparing the sterile tacrolimus nano suspension eye drops, homogeneously dispersed sterile nano eye drops which do not contain the organic solvent can be obtained, sterile operation steps are obvious reduced, the tacrolimus nano suspension eye drops prepared by adopting the method provided by the invention is sterile and qualified, all the inspection indexes are respectively consistent with those of the original product, and especially particle size and particle size distribution are highly consistent with those of the original product.