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The excess accumulation of nonessential Al3+ in the human body can cause serious health problems, thus there is significant interest in the detection of Al3+. A new ratio fluorescent probe (RF‐probe) based on the mixing of red‐emitting D‐penicillamine (DPA) stabilized copper nanoclusters (DPA‐CuNCs) with pyridoxal 5'‐phosphate (PLP) was designed for sensitive and selective determination of Al3+. The −CHO group of PLP is expected to form an imine linkage with the free −NH2 group available of the DPA on the surface of DPA‐CuNCs. The content of Al3+ could be mensurated by the as‐prepared RF‐probe with a linear response in the range of 1.33–46.67 μmol ⋅ L−1, and corresponding limit of detection (LOD) was calculated to be 0.33 μmol ⋅ L−1. Moreover, the RF‐probe was successfully extended to the assay of Al3+ in aluminum carbonate chewing tablets, baking powder, pond water and drink water with satisfactory results compared to the standard atomic absorption spectroscopy (AAS). [ABSTRACT FROM AUTHOR]