Connective tissue grafts for gingival recession treatment present significant challenges as they require an additional surgical site, leading to increased morbidity, extended operative times, and a more painful postoperative recovery for patients. Gelatin contains the arginine–glycine–aspartic acid (RGD) sequence, which supports cell adhesion and interactions. The development of gelatin hydrogels holds significant promise due to their biocompatibility, ease of customization, and structural resemblance to the extracellular matrix, making them a potential candidate for gingival regeneration. This study aimed to assess the physical and biological properties of crosslinked gelatin hydrogels using EDC/NHS with two crosslinker concentrations (GelCL12 and GelCL24) and compare these to non-crosslinked gelatin. Both groups underwent morphological, rheological, and chemical analysis. Biological assessments were conducted to evaluate human gingival fibroblast (HGF) proliferation, migration, and COL1 expression in response to the scaffolds. The crosslinked gelatin group exhibited greater interconnectivity and better physical characteristics without displaying cytotoxic effects on the cells. FTIR analysis revealed no significant chemical differences between the groups. Notably, the GelCL12 group significantly enhanced HGF migration and upregulated COL1 expression. Overall, GelCL12 met the required physical characteristics and biocompatibility, making it a promising scaffold for future gingival tissue regeneration applications. © 2024 by the authors.