<p>An optimal formulation of thermosensitive and injectable alginate-based hydrogels was prepared for loading therapeutic agents in drug delivery. Four constituents, including hydroxypropyl methylcellulose (HPMC), sodium alginate (SA), beta-glycerol phosphate (β-GP), and calcium chloride (CaCl₂), were used to obtain the optimal formulations. A surface response methodology (RSM), namely Box–Behnken, was employed, and based on the gelation temperature, two optimal hydrogel formulations were identified. Rheology, chemical and functional groups, morphology, biodegradability, swelling, and hydrogel biocompatibility were tested. Furthermore, menstrual blood-derived mesenchymal stem cell exosomes (Mens-exo) were used as a model drug to demonstrate the potential of the optimum hydrogels for carrying and releasing therapeutic agents. Two hydrogel formulations with gelling temperatures of 35 °C (H1) and 37 °C (H2) were selected for the relevant tests. The obtained storage modulus (G′) and loss modulus (G″) for gelling temperature, time, strain, and frequency tests showed that H1 hydrogel has more favorable rheological properties. Furthermore, in evaluating degradability at pH 6.5, H1 hydrogel was degraded for a longer time (154 h) and was more stable than H2 (100 h). Cells loaded in hydrogels indicated to superior biocompatibility of H1 hydrogel rather than H2. Moreover, the Mens-exo loading in H1 hydrogel exhibited a sustained release with reasonable degradability of the hydrogel. The optimal hydrogels were thermosensitive and injectable. In particular, the H1 hydrogel (SA = 0.889, HPMC = 2, β-GP = 5 and CaCl₂ = 3.306) showed high potential for loading therapeutic compounds.</p> Graphical Abstract <p></p>