A novel insulin composite delivery system was prepared and characterized. The composite consisted of a pH- and temperature-sensitive hydrogel, which is an oligomer serine-b-poly(lactide)-b-poly(ethylene glycol)-b-poly(lactide)-b-oligomer serine (OS-PLA-PEG-PLA-OS) pentablock copolymer, as matrix and chitosan-insulin electrosprayed nanospheres (CIN) as constituent materials. The properties of the OS-PLA-PEG-PLA-OS pentablock copolymer and the chitosan-insulin nanoparticles were characterized. The chitosan-insulin nanospheres uniformly distributed in the matrix had a reinforcing effect on the mechanical properties and prolonged the degradation time of the hydrogel depot under body conditions. The composite solutions accommodating different concentrations of the chitosan-insulin nanospheres were subcutaneously injected into induced diabetic BALB/c mice to study the in vivo insulin-release profile. The result showed that insulin concentrations in blood plasma were maintained at a steady-state level. Furthermore, the bio-properties of the insulin were retained and it showed a blood glucose level reducing effect for more than 60 hours after injection to a streptozotocin (STZ)-induced diabetic mouse model. The results suggested that this injectable pH-temperature sensitive hydrogel containing chitosan-insulin electrosprayed nanosphere composites has promising potential applications for type 1 diabetes treatment.