Cytoglobin (Cygb), a member of globin family, is a hemeprotein which was discovered 15 years ago. It contains a prosthetic group heme, which can reversibly bind an oxygen molecule between the iron ion of the porphyrin ring and a histidine of the polypeptide chain and play an important role in storing and delivering oxygen. There are two cysteines in C38 and C83 site of Cygb. Modification of the cysteine on Cygb can affect the oxygen-binding function of Cygb. In this article, chemical reagent 4, 4’- dithiodipyridine (4-PDS), N-ethylmaleimide (NEM), Oxidized Glutathione (GSSG) and Dithiothreitol (DTT) were used for modifying Cygb to intramolecular disulfide bond (Cygb-SS), thioether bonds (Cygb-SC), intermolecular disulfide bond (Cygb-SSG)and free sulfhydryls (Cygb-SH) respectively. The modified effect and product yield were detected by 4, 4’-dipyridine disulfide (4-PDS) spectroscopy method which can calculate modified rate by measuring the free sulfhydryl content in modified Cygb. The concentration of sulfhydryl in Cygb-SS and Cygb-SC samples is lower than one-tenth of the concentration of Cygb, indicating that the free sulfhydryl on the Cygb has been occupied by 4-PDS and NEM; The concentration of sulfhydryl in Cygb-SSG sample is comparable to that of the protein, indicating that one sulfhydryl in Cygb molecule participated in the reaction. Due to the steric hindrance of Cygb, the other sulfhydryl remained unchanged in the free sulfhydryl state; Double the concentration of sulfhydryl in the Cygb-SH sample as compared to the concentration of the Cygb indicates that one Cygb molecule contains two free sulfhydryl. We can know the modified yield by detecting the free sulfhydryl content of the four Cygbs respectively. The results showed that the four chemical reagents successfully modified cysteines on Cygb, and that the product yield reached more than 90%. This article measures the modified effect of Cygb by 4-PDS spectroscopy method and verifies the feasibility of this method by cysteine. In summary, the 4-PDS spectrometry method is accurate and reliable, which is complementary to the classical Ellman’s reagent method, and is more suitable for determining the content of sulfhydryl of compounds with absorption peak at 410~420 nm.