Tacrolimus (FK506) is widely used in ocular diseases such as corneal transplantation-host disease, uveitis, conjunctivitis, and dry eye disease (DED). However, its low aqueous solubility and poor ocular retention pose challenges for its application in the eye diseases. This study developed a novel FK506-loaded maleimide-functionalized cationic niosomes (FK506 M-CNS), aiming to prolong the retention time of FK506 in the eye and enhance its therapeutic efficacy. FK506 M-CNS had a particle size of 87.69 ± 1.05nm and zeta potential of 22.06 ± 1.01 mV. Results of histological evaluation through H&E staining and in vitro cytotoxicity of human corneal epithelial cells consistently revealed the excellent biocompatibility of FK506 M-CNS. FK506 M-CNS exhibited superior ocular retention compared to the market product Talymus®. FK506 M-CNS significantly alleviated the symptoms of DED and promoted the recovery of corneal epithelia. FK506 M-CNS group had the lowest expression levels of inflammatory factors associated with DED. These superiorities might be due to the electrostatic interaction between cationic niosomes and negatively charged mucin in the eye, and the covalent binding of maleimide with the thiol group in the mucin. The maleimide group improved the ocular retention and efficacy of FK506, but did not increase the toxicity. Results indicated that FK506 M-CNS had great potential as a nanopharmaceutical in the treatment of ocular diseases, and M-CNS could be a promising drug carrier for ophthalmic drug delivery systems.