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Constructed on the benzothiazole-oxanthracene structure, a fluorescent probe RBg for Cu⁺ was designed under the ESIPT mechanism and synthesized by incorporating amide bonds as the connecting group and glyoxal as the identifying group. Optical properties revealed a good sensitivity and a good linear relationship of the probe RBg with Cu⁺ in the concentration range of [Cu⁺] = 0–5.0 μmol L⁻¹. Ion competition and fluorescence-pH/time stability experiments offered further possibilities for dynamic Cu⁺ detection in an aqueous environment. HRMS analysis revealed a possible 1:1 combination of RBg and Cu⁺. In addition, colorimetric Cu⁺ detection and lysosome-targeted properties of the probe RBg were analyzed through RBg-doped PVDF nanofiber/test strips and RBg-Mito/Lyso trackers that were co-stained in living HeLa cells, enabling the probe’s future applications as real-time detection methods for dynamic Cu⁺ tracking in the lysosomes and Cu⁺ detection under diversified conditions.