Ozone (O3) has been widely used for reducing bacteria in fresh meat. However, O3 treatment has a negative impact on the red meat color, and the action mechanism of O3 on red meat color is still lack of research. The existence of myoglobin (Mb) is the basis for determining the key factors of red meat color. Therefore, the spectroscopic characteristics of myoglobin (Mb) under O3 were analyzed by UV-Vis absorption spectroscopy, fluorescence spectroscopy and circular dichroism (CD) spectroscopy. Moreover, the protein oxidation characteristics and molecular dynamics simulation were used to explore the effect and mechanism of O3 on Mb molecule. The results of spectroscopic studies show that the O3 treatment can decrease the intensity peak of the iron porphyrin ring at about 412 nm and the characteristic peak of oxygenated myoglobin (OMb) near 540 and 580 nm in the ultraviolet-visible spectrum of Mb. The characteristic peak of the iron porphyrin ring blue-shifted. It also caused changes in the endogenous fluorescence and synchronous fluorescence spectra of Mb measured at a fixed excitation wavelength of 280 nm, indicating that the fluorescence intensity of Mb was reduced by O3 and the fluorescence peak intensity contributed by the iron porphyrin group was increased and it also caused a blue shift in the characteristic peak of the fluorescence spectrum of the tyrosine residue. The characteristic peak intensity of the three-dimensional fluorescence spectrum decreased and the light scattering intensity increased. It was concluded that O3 would cause the protein oxidation of Mb, the exposure of hydrophobic group of the amino acid residues in Mb and the conformation change of the protein. The CD spectroscopy results show that the longer the contact time between O3 and myoglobin, the more obvious the change of protein secondary structure, resulting in a decrease in the content of α-helix and an increase in random curl. Combined with the chemical detection on the content and characteristics of Mb, it shows that O3 caused the decrease of OMb content, and the increase of MMb, carbonyl and sulfhydryl content, indicating that O3 treatments could lead to the protein oxidation. Moreover, O3 treatments increased the hydrophobicity of protein surface, indicating it resulted in the polarity change of the microenvironment of the protein system. Molecular dynamics simulation results show that O3 can increase the RMSD value of Mb peptide chain, affect the stability of Mb peptide chain, and weaken the interaction between porphyrin ring and Mb peptide chain. The change in RMSF value Mb peptide chain discovered that amino acid residues of Mb near the active pocket changed obviously; Molecular dynamics simulations of protein structural changes were consistent with the results of spectroscopic experiments, namely, the alpha-helix in Mb decreased and the irregular curl increased after O3 treatment. In conclusion, O3 treatment could interact with the residues of Mb, led to the changes in the secondary structure and the hydrophobicity of Mb, and brought on the oxidation of protein and the exposure of iron porphyrin ring, therefore resulting in the change of red meat color. This study can provide theoretical basis for the color protection of red meats.