Gene therapy has emerged as a promising approach for treating inner ear disorders, such as hearing loss and balance impairments, which require precise and efficient delivery mechanisms. This study introduces a dual-component delivery system combining RH-PAMAM G2 dendrimer-based polyplexes with hexanoyl glycol chitosan (HGC) thermogel to enhance gene delivery to the inner ear. The RH-PAMAM G2 dendrimers, modified with histidine and arginine, demonstrated high DNA binding affinity, low cytotoxicity, and effective cellular uptake, facilitating stable plasmid DNA (pDNA) transfection in HEI-OC1 auditory cells. Encapsulating these polyplexes within the HGC thermogel, an injectable and thermosensitive hydrogel, resulted in a supportive matrix that protects against premature clearance and provides sustained gene release upon intratympanic administration. In vivo studies in a mouse model confirmed substantial gene expression in the cochlear tissues, with widespread distribution in regions including the spiral ganglion and organ of Corti, compared to polyplexes alone. The HGC thermogel also exhibited favorable biocompatibility, with no observed inflammation or adverse effects in the middle ear tissues. This novel polyplex-HGC thermogel system demonstrates potential as a safe, efficient injectable gene delivery platform, offering a significant advance in gene therapy for inner ear disorders.