Excessive accumulation of mitochondrial reactive oxygen species (ROS) triggers the vicious circle in development of environment-induced dry eye disease (DED). Thus, mitochondria-specific antioxidants that in situ scavenge ROS and restore homeostasis in mitochondria can be employed to inhibit or alleviate environment-induced DED. Herein, novel mitochondria-specific anti-oxidative nanoparticles (NPs) consisting of negatively charged hyaluronic acid and the positively charged MitoQ were prepared by charge-driven self-assembly strategy assisted by heat treatment. The effects of operation temperature on size, morphology, zeta potential, and polydispersity index (PDI) of the MitoQ NPs were investigated. The NPs prepared by this strategy exhibited relatively higher drug encapsulation capacity and loading efficiency compared to that prepared from amphiphilic block copolymer using the traditional self-assembly method. The heat treatment enhanced the interaction between the charged molecules HA and MitoQ, thus the resultant NPs had more compact structure with uniform spherical morphology. In comparison with free MitoQ, MitoQ NPs had increased mitochondrial distribution, thereby exhibited a stronger mitochondrial ROS scavenging activity in the tested cell line. MitoQ NPs also exerted a better curative effect against environment-induced DED: it better alleviated the DED symptoms, reduced the ROS accumulation on eye surface, and enhanced the suppression of gene expression of inflammation related-factors in vivo. These results present the potential use of MitoQ NPs, for the treatment of a wide range of ROS-related diseases.