In this paper, the structural changes of myoglobin after salt treatment and the binding mode of substrate and pepsin/trypsin were studied by ultraviolet absorption spectroscopy, synchronous fluorescence spectroscopy, LC-MS/MS and molecular dynamics simulation. NaCl treatment changed the heme structure and hydrophobic cavity of myoglobin, which in turn decreased the digestibility of the protein. Trypsin was more effective for digestion of high molecular weight peptides. Molecular dynamics simulation showed that the binding of myoglobin and pepsin/trypsin was mainly maintained by hydrogen bond and hydrophobicity. In addition, the catalytic active center of pepsin is not suitable for long-time existence of low-molecular weight peptide under salt treatment. The findings provide a new insight into the low bioavailability of myoglobin.