The process of sludge bioleaching was studied by using pure H2S and gaseous sulfur matrix such as odor H2S as the substitute matrix of elemental sulfur. The effects of gaseous sulfur substrate on sludge acclimation, pH, ORP, microbial diversity, EPS infrared spectrum, Cr removal rate, and sludge dewatering performance were discussed. The results show that the intervention of H2S could significantly accelerate the decline rate of pH value in the process of sludge acclimation, reduce the dilution curve position of reaction system samples, increase the relative abundance of core bacteria in bioleaching, then T.t and T.f bacteria dependent on sludge leaching become the dominant bacteria. The addition of H2S is conducive to the rapid increase of ORP value in the process of sludge bioleaching; it can improve the adsorption and chemical conversion rate of heavy metals by the core chemical groups in the process of sludge bioleaching, and improve Cr removal rate. When the leaching is carried out to 10 days, the Cr removal rate of the S+pure H2S reaction group is 22.8% and 16.2% higher than that of the other two groups, respectively. Bioleaching can reduce the specific resistance of sludge and improve the centrifugal dewatering rate of sludge. In the reaction system with H2S as the substitute of sulfur matrix, the dosage of elemental S(sulfur powder) can be reduced by 50%, and the reaction effect is better than that of traditional matrix. To a certain extent, the H2S in the odor of sewage treatment plant can replace pure H2S.