AbstractPollution of heavy metals in agricultural environments is a growing problem to the health of the world’s human population. Green, low-cost, and efficient detection methods can help control such pollution. In this study, a protein biosensor, mApple-D6A3, was built from rice-derived Cd²⁺-binding protein D6A3 fused with the red fluorescent protein mApple at the N-terminus to detect the contents of heavy metals. Fluorescence intensity of mApple fused with D6A3 indicated the biosensor’s sensitivity to metal ions and its intensity was more stable under alkaline conditions. mApple-D6A3 was most sensitive to Cu²⁺, then Ni²⁺, then Cd²⁺. Isothermal titration calorimetry experiments demonstrated that mApple-D6A3 successfully bound to each of these three metal ions, and its ability to bind the ions was, from strongest to weakest, Cu²⁺ > Cd²⁺ > Ni²⁺. There were strong linear relationships between the fluorescence intensity of mApple-D6A3 and concentrations of Cd²⁺ (0–100 μM), Cu²⁺ (0–60 μM) and Ni²⁺ (0–120 μM), and their respective R² values were 0.994, 0.973 and 0.973. When mApple-D6A3 was applied to detect concentrations of heavy metal ions in water (0–0.1 mM) or culture medium (0–1 mM), its accuracy for detection attained more than 80%. This study demonstrates the potential of this biosensor as a tool for detection of heavy metal ions.