The herein work elucidated colloid performances of thermosensitive in situ-forming ophthalmic gel established by poloxamer 407 (F127), mainly including facile and simple methods (load-bearing method, tilting plate method, and eye simulation method) to analyze and evaluate F127 gel and intensively addressed capacity of F127 gel for loading drugs. The results demonstrated that the poorly water-soluble berberine hydrochloride improved entangled intensity of F127 and therefore reduced the sol dynamic rate, which contributed to lower gelation temperature and enhancement of cornea adhesion, further lowering the rate of gel corrosion together with drug release. Importantly, the addition of hydroxy propyl methyl cellulose (HPMC) K15M contributed to stronger gel strength because strong interaction forces were formed between HPMC K15M and F127 when the molecular chain segments of HPMC K15M interspersed among F127 network. All the aforementioned results provide valuable instruction for designing and evaluating thermosensitive in situ-forming ophthalmic gel.