Despite considerable progress having been made in thermosensitive protein hydrogels, regulating their thermal transitions remains a challenge due to the intricate molecular structures and interactions of the underlying protein polymers. Here we report a genetic fusion strategy to tune the unique dual thermal transitions of the C-terminal domain (CTD) of spider major ampullate spidroin 1, and explore the regulation mechanism by biophysical characterization and molecular dynamics simulations. We found that the fusion of elastin-like polypeptides (ELPs) tuned the dual transition temperatures of CTD to a physiologically relevant window, by introducing extra hydrogen bonding at low temperatures and hydrophobic interactions at high temperatures. The resulting hydrogels constructed from the fusion proteins were demonstrated to be a promising vehicle for cell preservation and delivery. This study provides insights on the regulation of the dual thermosensitive protein hydrogels and suggests a potential application of the hydrogels for consolidated cell storage and delivery.