Objective In order to improve the problems of poor water-solubility and low bioavailability of ocular tacrolimus, a cationic nanoemulsion in-situ gel of tacrolimus was developed and its drug release behavior in vitro was studied to provide a basis for further research. Methods Tacrolimus-loaded cationic nanoemulsion was prepared by high pressure homogenization and dispersed in poloxamer 407 and poloxamer 188 to prepare tacrolimus-loaded cationic nanoemulsion-based in-situ gel. The membraneless dissolution model was used to study the in vitro drug release behavior. Results The inverse glass bottle method was used to determine the gelation temperature. The optimal formulation of gel matrix was screened out as 26% poloxamer 407 and 12% poloxamer 188. The in vitro release results showed that the rate of gel dissolution determined the in vitro drug release. Conclusion Tacrolimus-loaded cationic nanoemulsion-based in-situ gel is successfully prepared. Its in vitro drug release is stable. It meets the requirement of ophthalmic formulation and shows good prospects for ocular application.