ABSTRACT: Objective To prepare ocular levofloxacin hydrochloride nanoparticles thermo-sensitive in situ gels(LVFX-NPs-TISG)and to study its drug release in vitro. Methods Ocular levofloxacin hydrochloride nanoparticles were prepared by ionic cross-linking method. Poloxamer 407 and 188 were used as the thermo-sensitive material. A central composite design-response surface method(CCD-RSM)was adopted to optimize formulation of gel by measuring gelation temperatures with and without artificial tears. A release method was used to study the drug release in vitro. Results The mean diameter of drug loaded nanoparticles was(60.7±5.1)nm. The encapsulation efficiency and drug loading were(62.5±1.8)% and(10.43±0.30)%. The optimized formula was selected as poloxamer 407:poloxamer 188(22%:6%)and the gelation temperature was(32.3±0.2)℃. Within 24 hours, accumulative release of levofloxacin hydrochloride was 71.9%. Conclusions LVFX-NPs-TISG showed the advantages of nanoparticles and in situ gels with the properties of desired gelation temperature and sustained drug release in vitro, which hopefully provides a promising ophthalmic drug delivery of levofloxacin hydrochloride.