To achieve the regulation of spatial crosslinking degree of gelatin solution system and enhance its gelling properties, addressing the issues of poor gelation and low recycling rates during the resource utilization of gelatin solutions, choline chloride-based deep eutectic solvents (DES) were used to regulate the protein conformation in the gelatin solution system. Using gelatin solution as a control group, five different acidic and alkaline choline chloride-based DES were introduced. The effects of DES acidity on the gelling behavior of gelatin solutions were investigated through transmittance ratio, particle size and Zeta potential measurements, and UV-Vis, FTIR, XPS, circular dichroism spectroscopy characterization, and thermal performance testing. Results indicated that acidic DES (choline chloride + oxalic acid, choline chloride + p-toluenesulfonic acid) mainly exerted a pH effect on gelatin solutions by altering the chemical environment, reducing chemical structure strength and stability. Neutral DES (choline chloride + glycerol, choline chloride + sorbitol) disrupted the original hydrogen bonding network within gelatin, forming a new hydrogen bonds. Under the influence of the choline chloride-glycerol deep eutectic solvent, the total content of α-helix and β-sheet structures in the proteins increased from 44.30% to 70.86%, leading to a more ordered and rigid structural transition, indirectly increasing the folded conformations in the tertiary structure, and altering the spatial cross-linking network, thereby enhancing the gelling action of the gelatin solution.