A series of mixed hydrogels of PLGA-PEG-PLGA and PCLA-PEG-PCLA were synthesized, and investigated in terms of their critical micelle concentration, stability and thermosensitive properties. Also, some mixed hydrogel was selected to prepare Depot-gel-in-Ms-in-Matrix-gel system for the treatment of type 2 diabetes mellitus. Briefly, Exenatide (EXT) loaded hydrogels was encapsulated in PLGA microspheres (Ms) and further encapsulated into blank hydrogel. The mechanism of Exenatide release involved drug diffusion, hydrogel diffusion, PLGA erosion and mixed hydrogel erosion. The results showed that EXT release in vitro was at a sustained rate for 46 days, because it is controlled by the inner-deport-gel, the Ms matrix and the outer-Matrix-gel successively. No burst release or platform was observed due to the interception function and control function of the outer-Matrix-gel. The biological activity of EXT was protected, because the hydrophilic EXT molecules tend to distribute in the hydrophilic domain of the mixed hydrogel. In vivo, a single injection of Depot-gel-in-Ms-in-Matrix-gel allowed mice to maintain a stable blood glucose concentration and well-controlled body weight for 20 days. In addition, results of oral glucose tolerance test and Hematoxylin-Eosin staining demonstrated that triple-barrier Depot-gel-in-Ms-in-Matrix-gel was a promising hydrophilic protein/polypeptide-loaded long-acting system with high drug bioactivity.