In order to analyze the factors affecting yolk blackening in salted duck eggs, this study investigated the differences in the color difference, metal ion contents and proteomics in different parts of normal and black-yolked salted duck eggs. The effects of these factors on the degree and rate of yolk blackening were studied by single factor experiments. The results showed that the higher the contents of Fe and Cu, the smaller the color difference value of yolk and the blacker the color. It was speculated that the sulfide formed by the combination of Fe2+ in phosvitin (Pv) and S2- was responsible for the blackening of egg yolk, and the degradation and desulfurization of cysteine was an important contributor to sulfur production in egg yolk. Microbial load, temperature and pH were important factors affecting yolk blackening. With increasing bacterial load and curing temperature of duck eggs, the rate of yolk blackening increased. H2S was produced in the pH range of 7.0–8.0, and the gas production rate was the fastest at pH 7.5. In summary, it can be inferred that yolk blackening in salted duck eggs is caused by the discoloration reaction between sulfur and iron elements in egg yolk. Curing temperature, pH and the bacterial load of duck eggs have a great influence on yolk blackening.