The targeted recognition of aluminum ions (Al3+) is indispensable owing to their significant environmental abundance and possible health effects. A crucial prerequisite for their practical use in biological systems and environmental monitoring is the advancement of sensitive and selective fluorescence sensors for Al3+ that works effectively in aqueous conditions. Herein, we synthesize a new Schiff base ligand (L) based on isoniazid as a highly sensitive and selective turn-on fluorescent probe for detection of Al3+ in DMSO-water (0.1:9.9, v/v) solutions. A key novelty of this work lies in the effective performance of the probe in a water-rich medium. When the probe binds to Al3+, it shows a noticeable increase in fluorescence, with an emission maximum at 467 nm and excitation wavelength of 330 nm. The binding constant was found to be 0.17 x 104 M- 1, and the detection limit was 53.4 nM, which is significantly lower than the permissible grade for drinking water of WHO. Comprehensive analytical and computational studies validated the 1:2 binding stoichiometry and elucidated the sensing mechanism, which involves suppression of cis-trans isomerization and chelation-enhanced fluorescence (CHEF). Its potential use in real-time biological monitoring was further highlighted by bioimaging tests conducted on HepG2 cells, which showed that the probe L could effectively pass through the cellular membrane and preferentially visualize intracellular Al3+ ions.