Monitoring heavy metal pollution in riverine ecosystems requires a comprehensive understanding of their distribution, bioavailability, and assimilation into aquatic biota, including potential risks to humans. Macroinvertebrates provide valuable insights into metal contamination; this study investigates metal assimilation in rivers by the caddisfly larva Cheumatopsyche sp. We hypothesized that heavy metals derived from water and sediments, largely originating from anthropogenic activities, are assimilated and reflected through the expression of metallothionein (MT) in Cheumatopsyche sp. Accordingly, this study aimed to evaluate the relationship between metal bioavailability and MT responses as a molecular biomarker. Field sampling was conducted at five ecologically representative locations distributed along the longitudinal gradient of the Bone River watershed, spanning from the upper reach to downstream. Heavy metals were analysed using the atomic absorption spectrophotometry method, while MT density and intensity were assessed through immunohistochemistry. The research revealed significant spatial variation in both heavy metal concentrations and MT expression across stations. Metal levels in water and sediment were strongly correlated with bioaccumulation in biota. Cadmium (Cd) exhibited the highest bioavailability, whereas generalized additive models (GAMs) identified mercury (Hg) as the primary determinant of MT density and intensity, with lead (Pb) influencing intensity only, and Cd showing no significant effect. Collectively, our findings demonstrate that Cheumatopsyche sp. larvae effectively trace the mobilization and distribution of contaminants in aquatic systems, underscoring their potential as bioindicators for ecosystem management in polluted rivers.