Cyclin D1 has become a potential target for anti-tumor therapy. Recently, a novel human anti‑cyclin D1 single‑chain variable fragment (AD5) was identified, which demonstrated specific binding activity to cyclin D1 and exhibited anti‑tumor effects. However, the detailed characteristics of AD5 remain unclear. In the present study, the structure and activity of AD5 in the presence of copper II (Cu2+) or iron III (Fe3+) metal ions was investigated by fluorescence spectroscopy, synchronous fluorescence and enzyme‑linked immunosorbent assay. Cu2+ and Fe3+ were able to bind to AD5 and quench the fluorescence intensity of AD5 primarily by static quenching, which slightly altered the conformation of AD5 at temperatures of 293, 298 and 303 K; however, these temperatures demonstrated different effects on the activity of AD5. These results may be of value for the clinical application of anti-cyclin D1 single chain antibodies in the future.