Abstract: In the work, porcine hemoglobin (PHb) was applied to prepare porcine nitrosohemoglobin (PHbNO), and basic structures and properties of PHb were first characterized by differential scanning calorimetry, UV-Vis spectrum and potential analysis, and then the structural stability and mechanism of PHbNO were studied by dialysis method. The results from analyzing the raw materials PHb indicated that there were no obvious differences in the composition, purity, thermal stability and UV-Vis spectra characteristics by comparing with the standard PHb; the results from UV-Vis showed that porphyrin structure in PHbNO without or in partial dialysis was significantly changed; fluorescence spectroscopic analysis also suggested that structure and microenvironment around tryptophan or tyrosine residues in PHbNO without or in partial dialysis were significantly altered; the results from infrared and circular dichroism indicated that secondary structures of PHbNO changed obviously, however, four spectral characteristics of PHbNO after dialyzing thoroughly to remove small molecules were basically the same as those of PHb. Therefore, it could be inferred that nitrosylation of PHb was reversible, and stability of PHbNO depended on NaNO₂, and it could be deduced by comprehensive analysis that the stability and mechanism of PHbNO was that NO generated in acidic solution by NaNO₂ could react with Fe²⁺ in PHb molecule by a reversible style to generate PHbNO, so the stability of PHbNO depended on the reactants. The study could provide a theoretical basis and data for the further study and application of PHbNO.