Non‑antibody‑binding proteins (nABPs) have attracted attention in the field of alternative antibody approaches due to their ability to penetrate cell membranes. CD105 has been demonstrated to be a useful marker for identifying angiogenic endothelial, mesenchymal stem and osteosarcoma cells. In the present study, a novel non‑antibody‑binding protein (namely nABP296) with a high affinity for recombinant human CD105 was identified by M13 phage biopanning. Fluorescence microscopy, flow cytometry and in vivo animal imaging were used to confirm the visualization effect of the novel peptide to the CD105‑positive MNNG/HOS cell line in vivo and in vitro. Immunofluorescence of tissue sections was also used to identify the target efficiency of the peptide in tumour sections derived from an MNNG/HOS xenograft tumour model and osteosarcoma patients. This peptide specifically was found to bind to the CD105‑positive osteosarcoma MNNG/HOS cell line and the osteosarcoma cells in the histological sections derived from an MNNG/HOS xenograft model and osteosarcoma patients in vitro. This peptide also successfully labelled an animal osteosarcoma xenograft model in vivo. Taken together, a novel peptide that can be used as a potent agent for CD105 molecular targeting was identified, and this peptide can be applied for osteosarcoma visualization in vitro and in vivo.