The present study, COLL: AV: GPO bioactive scaffold prepared from collagen (COLL), Aloe vera (AV) and graphene oxide (GPO) for wound healing application. Bioactive scaffolds were examined by Fourier Transform Infrared Spectroscopy (FT-IR), Thermogravimetry Analysis (TGA) to understand the intermolecular interaction and their influence on the thermal property of the composite. Scanning Electron Microscope (SEM) image of the bioactive scaffold showed porous and smooth surface. Atomic Force Microscopy (AFM) observed fibrils with intermittent banding patterns. Bioactive scaffold possessed tensile strength (MPa), elongation at break (%), and water absorption properties (%). Bioactive scaffold exhibited good anti-bacterial activity against E.coli and S. aureus. MTT assay on the composites showed enhanced cell growth of human keratinocyte cell line (HaCaT) without exhibiting any toxicity. A higher rate of cell growth present in the COLL: AV: GPO bioactive scaffold compared to the other samples (COLL and COLL: AV) was observed. Acceleration of wound healing in COLL: AV: GPO bioactive scaffold treated rats were significantly in the in vivo experiments. These results suggested that the COLL: AV: GPO bioactive scaffold could be promising materials from wound healing applications.