Heavy metal pollution in freshwater ecosystems is a critical issue because it threatens the ecosystem as well as public health. Early detection of these pollutants is therefore essential, and biomarker analysis can be an ideal way to achieve this. Metallothionein (MT) protein is a widely-used molecular biomarker related to the physiological and biological changes which suitable bioindicators, like freshwater snails, undergo in stressful environments. The purpose of this research is to assess the relationship between the heavy metals lead (Pb), cadmium (Cd) and mercury (Hg) and MT levels in freshwater snails (Sulcospira testudinaria) in the Brantas River watershed. Heavy metals were assayed using an atomic absorption spectrophotometer, while MT levels were analysed using indirect enzyme-linked immunosorbent assay (ELISA). Water quality parameters including temperature, dissolved oxygen (DO), biological oxygen demand (BOD), ammonia concentration, and phenol concentration were also measured. Samples were obtained from ten sampling sites at Brantas River watershed. The results indicated that Pb concentration ranged from 0.001 – 0.006 mg/l, Hg from 0.001 – 0.005 mg/l, and Cd from 0.005 - 0.03 mg/l, while MT concentration ranged from 0.40 - 0.80 ng/g. Relationship analysis between heavy metals and MT level in this study revealed the significant effect of Pb concentration on MT values in Sulcospira testudinaria. Therefore, MT in this snail is a potential biomarker for Pb contamination.