A simple, rapid, and inexpensive method for the synthesis of cyclic arginine-glycine-aspartic acid (RGD) peptide conjugated gold nanoclusters (RGD-GNCs) was reported. The nanoclusters were synthesized with chloraurate as precursor and cyclic RGD peptides as both reducing and protecting agent directly under alkali condition, and the whole synthetic process only took 15 min at room temperature. The properties of the nanoclusters were characterized by means of ultraviolet–visible spectra, Fourier transform infrared spectroscopy (FTIR), fluorescence, transmission electron microscopy (TEM), and X-ray photoelectron spectroscopy (XPS). The prepared gold nanoclusters were successfully used as a contrast agent in fluorescence imaging of the melanoma A375 cells, which overexpress the integrin αvβ3. The results demonstrated that our nanoclusters possess good biocompatibility, stability, and low toxicity. Moreover, the method is expected to be applicable to the synthesis of nanoclusters conjugated with other biomolecules.