The development of effective wound dressings requires biomaterial platforms with structural integrity, biocompatibility, and multifunctional bioactivity. This study engineered composite hydrogels by blending tilapia skin-derived decellularized extracellular matrix (TSdECM) with Aloe vera (AV) at 0, 5, and 10 mg/mL for wound healing. TSdECM/AV-10 exhibited the highest porosity, water absorption, and swelling capacity, while retaining moderate degradability. Rheological analysis confirmed shear-thinning behavior and tunable storage modulus, with AV reducing viscosity. Low-temperature extrusion-based 3D printing at −20 °C yielded scaffolds with high shape fidelity. The constructs supported robust fibroblast viability and enhanced proliferation, endothelial tube formation, and epidermal differentiation. Air-liquid interface co-culture revealed upregulated cytokeratin 10 and 14, indicative of improved stratification. Macrophage polarization assays showed elevated M2 markers, reduced pro-inflammatory genes, and strong immunomodulatory effects. Moreover, TSdECM/AV-10 scaffolds exhibited potent antibacterial activity against Pseudomonas aeruginosa and promoted rapid blood clot formation. Overall, these findings highlight TSdECM/AV scaffolds as versatile, bioactive platforms that integrate mechanical resilience with pro-regenerative, immunomodulatory, and antimicrobial functions, offering strong translational potential for advanced skin tissue engineering.