To determine the presence of sulfhydryl groups on natural aquatic biofilms and their adsorption characteristics for typical heavy metals, a method for sulfhydryl masking in the biofilms based on a specific masking agent was established in this study. Based on this method, surface concentration of sulfhydryl group in natural biofilms and their adsorption characteristics for typical heavy metals, including Cu, Pb, and Cd, at different pH were investigated. The results indicate that the established masking method can effectively mask sulfhydryl groups and has little effect on microorganisms in biofilms. There are relatively low concentrations of sulfhydryl groups on the surface of natural biofilms, with a concentration of (5.8 ± 0.6) μmol/g, accounting for 5.7% of the total site concentration on the biofilms. Despite the low concentration of sulfhydryl groups, their stronger metal binding capacity makes them significantly contribute to metal adsorption when the metal concentration is low (the theoretical loading of heavy metals by the biofilm is less than 1.0μmol/g). This pattern is essentially unaffected by the pH of the adsorption system and the type of heavy metal. This proves that sulfhydryl groups in biofilms also have an important impact on the behavior and risk of heavy metals in natural aquatic environments with low metal content, further highlighting the environmental significance of natural biofilms.