Long-term injectable microspheres have some inherent disadvantages such as migration of microspheres from the original site and the burst effect. In order to avoid these problems, microsphere-loaded thermosensitive hydrogel system was designed and expected to achieve a zero-order release of biomolecular drugs in relative high initial drug loadings. Lysozyme, an antibacterial protein usually used to reduce prosthetic valve endocarditis,was selected as the model drug. Poly (DL-lactide-co-glycolide) (PLGA) microspheres, prepared by solvent evaporation method, were employed to encapsulate lysozyme and dispersed into thermosensitive pre-gel solution containing methylcellulose (MC), polyethylene glycol (PEG), sodium citrate (SC), and sodium alginate (SA). The mixture could act as a drug reservoir by performing sol-gel transition rapidly if the temperature was raised from room temperature to 37℃. The in vitro release results showed that the burst effect was avoided due to strengthening of diffusion resistance in the gel. The formulation was able to deliver lysozyme for over 30 days in a nearly zero-order release profile with a rate of 32.8μg·d-1 which exhibits its remarkable potential for effective application in long-term drug delivery.