In this article, the interaction mechanism of Ga(3+)-hypocrellin A (Ga(3+)-HA) with myoglobin (Mb) is studied in detail through various spectroscopic technologies. UV-vis absorption and fluorescence spectra demonstrate the interaction process. The Stern-Volmer plot and the time-resolved fluorescence study suggest the fluorescence quenching mechanism of Mb by Ga(3+)-HA is a static quenching procedure, and the electronic transfer forces play a major role in binding Ga(3+)-HA to Mb. Furthermore, synchronous fluorescence studies and circular dichroism (CD) spectra reveal that the conformation of Mb is changed after its conjugation with Ga(3+)-HA.