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A simple and rapid As³⁺ detection method using 3-nitro-L-tyrosine (N-Tyr) is reported. We discovered the specific property of N-Tyr, which specifically chelates As³⁺. The reaction between As³⁺ and N-Tyr induces a prompt color change to vivid yellow, concomitantly increasing the absorbance at 430 nm. The selectivity for As³⁺ is confirmed by competitive binding experiments with various metal ions (Hg²⁺, Pb²⁺, Cd²⁺, Cr³⁺, Mg²⁺, Ni²⁺, Cu²⁺, Fe²⁺, Ca²⁺, Zn²⁺, and Mn²⁺). Also, the N-Tyr binding site, binding affinity, and As³⁺/N-Tyr reaction stoichiometry are investigated. The specific reaction is utilized to design a sensor that enables the quantitative detection of As³⁺ in the 0.1−100 μM range with good linearity (R² = 0.995). Furthermore, the method’s applicability for the analysis of real samples, e.g., tap and river water, is successfully confirmed, with good recoveries (94.32−109.15%) using As³⁺-spiked real water samples. We believe that our discovering and its application for As³⁺ analysis can be effectively utilized in environmental analyses such as those conducted in water management facilities, with simplicity, rapidity, and ease.