Myoglobin (Mb) made up of a multipeptides train and a heme prosthetic group is a kind of protein taking charge of O2 stock and distribution in mammal cells, especially in muscle cells. The heme-iron plays a key role in O2 transfer and transport. In the present paper, the direct interaction between heme-iron of myoglobin and additional metal ions [Cu (II), Zn (II) and Co( II)] was studied by UV-Vis spectra. It was found that heme-iron of myoglobin directly interacted with additional Cu(II), Zn(II) and Co(II), these metal ions could drag iron ion out from heme prosthetic group of myoglobin, and subsequently myoglobin became myoglobin derivatives lacking iron ion. At the same time, the effect of the additional metal ions concentration on the direction interaction was studied. It was shown that the direct interaction increased gradually with the amount of external metal ions added. When the ratio of Mb and metal ions is 1 : 10, the interaction intension between the three metal ions and Mb is Co(II), Zn(II) and Cu(II) in turn. For the first time, the authors confirmed that the direct interaction has occurred between heme-iron of myoglobin and additional metal ions, and saw about how the metal ions intension affects the direct interaction.