A new Schiff base tcph, derived from 2-thiophene carboxylic acid hydrazide, has been synthesized and characterized by various spectroscopic techniques. The molecular structure of the compound has also been determined by X-ray crystallography. The tcph acts as a selective Al3+ and F− induced OFF-ON-OFF type of probe in aqueous media. The 1:1 binding stoichiometry between probe and Al3+ has been established from Job’s plot and further supported by ESI–MS studies. The limit of detection of Al3+ ions is determined by 3σ methods, which is found to be 1.35×10−9M. The coordination environment for the tcph-Al3+ complex is delineated by NMR titration and DFT calculations. Detailed insights of probe–metal interaction mechanism have been studied by density functional theory (DFT) as well as time dependent–DFT calculations. MTT assay of the probe on live SiHa cells suggests no serious cytotoxicity in cells even at higher concentration. The probe tcph and its tcph-Al3+ complex have also been successfully applied to detect Al3+ and F− ions in living cells (SiHa cells), respectively.