Periodontal disease in dogs leads to progressive bone loss and adversely impacts overall health. However, cost-effective regenerative strategies are still limited in veterinary practice. This study aimed to develop and evaluate a novel tannic acid (TA)-gelatin-based hydrogel (Gel), incorporating graphene oxide (GO) and hydroxyapatite nanoparticles (HA), as a potential barrier material for guided tissue regeneration (GTR) applications. The hydrogels-Gel, Gel-GO, Gel-HA, and Gel-GO-HA-were characterized for chemical structure, molecular interactions, surface morphology, nanoparticle dispersion, and tensile strength. Cytotoxicity was assessed using L929 fibroblasts (ISO 10993-5), while cell viability/proliferation, morphology, and alkaline phosphatase (ALP) production were evaluated using canine periodontal ligament-derived cells. Results show that crosslinking with tannic acid enhanced the incorporation of graphene oxide and hydroxyapatite nanoparticles via hydrogen bonding into TA-gelatin-based hydrogels. This combination increased surface roughness, reduced degradation rate, and enabled shape memory behavior, critical for guided tissue regeneration (GTR) membranes. The extracts from Gel-HA-GO showed that cytotoxicity was both time- and concentration-dependent in L929 fibroblasts, whereas enhanced cell proliferation and increased ALP production were observed in cultures derived from canine periodontal ligament cells. These findings suggest that TA-gelatin-based hydrogels incorporating GO and HA demonstrated favorable mechanical and physicochemical properties, biocompatibility, and osteogenic potential. These attributes suggest their viability as a promising composite for the development of innovative GTR strategies to address periodontal tissue loss in veterinary medicine.