Due to their relatively large molecular sizes and delicate nature, biologic drugs such as peptides, proteins, and antibodies often require high and repeated dosing, which can cause undesired side effects and physical discomfort in patients and render many therapies inordinately expensive. To enhance the efficacy of biologic drugs, they could be encapsulated into polymeric hydrogel formulations to preserve their stability and help tune their release in the body to their most favorable profile of action for a given therapy. In this study, a series of injectable, thermoresponsive hydrogel formulations were evaluated as controlled delivery systems for various peptides and proteins, including insulin, Merck proprietary peptides (glucagon-like peptide analogue and modified insulin analogue), bovine serum albumin, and immunoglobulin G. These hydrogels were prepared using concentrated solutions of poly(lactide- co -glycolide)– block -poly(ethylene glycol)– block -poly(lactide- co -glycolide) (PLGA–PEG–PLGA), which can undergo temperature-induced sol–gel transitions and spontaneously solidify into hydrogels near the body temperature, serving as an in situ depot for sustained drug release. The thermoresponsiveness and gelation properties of these triblock copolymers were characterized by dynamic light scattering (DLS) and oscillatory rheology, respectively. The impact of different hydrogel-forming polymers on release kinetics was systematically investigated based on their hydrophobicity (LA/GA ratios), polymer concentrations (20, 25, and 30%), and phase stability. These hydrogels were able to release active peptides and proteins in a controlled manner from 4 to 35 days, depending on the polymer concentration, solubility nature, and molecular sizes of the cargoes. Biophysical studies via size exclusion chromatography (SEC) and circular dichroism (CD) indicated that the encapsulation and release did not adversely affect the protein conformation and stability. Finally, a selected PLGA–PEG–PLGA hydrogel system was further investigated by the encapsulation of a therapeutic glucagon-like peptide analogue and a modified insulin peptide analogue in diabetic mouse and minipig models for studies of glucose-lowering efficacy and pharmacokinetics, where superior sustained peptide release profiles and long-lasting glucose-lowering effects were observed in vivo without any significant tolerability issues compared to peptide solution controls. These results suggest the promise of developing injectable thermoresponsive hydrogel formulations for the tunable release of protein therapeutics to improve patient’s comfort, convenience, and compliance.