Bacterial keratitis is an eye infectious disease which became a global concern due to its impact to visual impairment in worldwide. Cefazolin (CFZ) is the first-line drug for the treatment of bacterial keratitis and only available in the drop dosage form. However, the administration of eye drops results in lack of bioavailability, which is below 5%. Therefore, an innovation is required in an attempt to overcome these problems. In this study, the development of a mucoadhesive-thermosensitive gel in situ preparation was carried out. A combination of Pluronic F127 and F68 was used as a thermosensitive agent. To increase the contact time, a mucoadhesive agent, namely hyaluronic acid was added to the formulation. Several steps of evaluation were performed to ensure that the developed formula possessed desired characteristics, including determination of gelation temperature, pH test, viscosity test, rheology test, mucoadhesive test, drug content test, in vitro drug release test, in vivo irritation test on experimental animals, ex vivo permeation test, and hemolysis test. The results that the formulations developed exhibited desired characteristics, with gelation temperatures of around 37 °C. The formulation could also control the release and improve the localization of CFZ in the ocular tissue compared to control solution. Furthermore, the incorporation of CFZ into this approach did not change the antimicrobial activity of CZ against Pseudomonas aeruginosa. Importantly, no toxicity and irritation were observed after the application of this approach. However, further research is needed to evaluate the pharmacokinetic and pharmacodynamic in the appropriate animal models.