In the current study, we reported a novel label-free and facile colorimetric approach for the sequential detection of copper ion (Cu2+), l-arginine (Arg), and l-cysteine (Cys) in the H2O (10.0mmolL(-1) HEPES buffer solution, pH7.0) using Reactive Blue 4 (RB4). First, the presence of Cu2+ led to a naked-eye color and spectral changes according to the binding site-signaling subunit approach. Then, the RB4-Cu2+ complex was successfully applied for Cys and Arg through different recognition pathways. The optical signals for Arg were observed due to its association involving the amino group, as well as the participation of the carboxylate group in a bidentate form to the complex, while selective behavior for Cys was explained by a metal displacement mechanism. The limits of detection for Cu2+, Arg, and Cys were calculated to be 1.96, 1.06, and 1.33molL(-1), respectively. It could also be employed for the determination of three analytes in environmental, biological, and pharmaceutical samples. Importantly, the test strips based on RB4-Cu2+ complex could be used as a solid-state sensor for the detection of Cys and Arg. In addition, NAND and IMPLICATION molecular logic gates were obtained by using chemical inputs and UV-Vis absorbance signal as the output.