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Peripheral nerve blockade is an important perioperative intervention for pain management. However, this management always results in a limited analgesia effect due to the rapid elimination of local anesthetic drugs after a single injection, while continuous infusion leads to multiple side effects associated with catheter insertion. In this study, three thermosensitive hydrogels, i.e., Pluronic F127, and poly(l-alanine)- block -poly(ethylene glycol)- block -poly(l-alanine) (PAla-PEG-PAla), and poly(lactic- co -glycolic acid)- block -poly(ethylene glycol)- block -poly(lactic- co -glycolic acid) (PLGA-PEG-PLGA), with distinct properties were applied to encapsulate bupivacaine (BUP) for prolonged analgesia with one single injection. All three hydrogel platforms exhibited a homogeneous three-dimensional structure with thermosensitive properties and high BUP encapsulation efficiency. The BUP loaded in PAla-PEG-PAla showed a sustained drug release profile, which was advantageous over those from Pluronic F127 and PLGA-PEG-PLGA with either burst release or inadequate release. The hydrogel platforms exhibited prolonged nerve blockade duration compared with BUP·HCl in vivo . Furthermore, the in vivo residence period of PAla-PEG-PAla/BFB was shorter than that of PLGA-PEG-PLGA/BFB while longer than that of Pluronic F127/BFB. All the hydrogels induced reversible inflammatory response without neurotoxicity. Overall, in comparison with the other two hydrogel platforms, PAla-PEG-PAla exhibited controlled drug release, appropriate residence period, and long-acting analgesia effect. Moreover, it might be a potential method to meet the different demands of regional nerve blockade and guide clinical pain management.