This manuscript introduced a new optically active chemosensor L containing rhodamine 6G as the fluorogenic unit and the vitamin B6 cofactor pyridoxal 5′-phosphate (PLP) as the metal ions binding unit. Sensor L showed a significant turn-on fluorescence at 550 nm selectively in the presence of Al3+ and Cr3+ ions due to the complexation prompted opening of the spirocyclic ring. Using sensor L, the concentration of Al3+ and Cr3+ ions can be detected down to 0.41 µM and 2.33 µM, respectively. Sensor L formed a metal complex in 1:1 binding stoichiometry in solution with the estimated binding constant of 11,310 M−1 and 8373 M−1 with Al3+ and Cr3+, respectively. The probable 3D structure and the electronic properties of L and its complexes were proposed by semi-empirical PM6 method. Further, the bioimaging experiments were performed to examine the potential of L to detect the intracellular Al3+ and Cr3+ ions present in the live HeLa cells. © 2019 Elsevier B.V.