Highly selective spectrofluorimetric detection of Fe3+ ions in acidic medium is developed using novel fluorescent sensor, namely 4-(2-(3-Methyl-5-oxo-1-tosyl-1H-pyrazol-4(5H)-ylidene) hydrazinyl)-N- (pyrimidin-2-yl) benzenesulfonamide (PHBS-sensor), which gives an selective fluorescence quenching response at λEmission 379 nm. The factors affecting fluorescence detection process, such as the pH, solvent type, buffer type, probe concentration, reaction time and co-existing ions were systematically studied and optimized. Theoretical modeling of Fe3+ monitoring through DFT calculation was investigated to display the mechanism of PHBS-Fe3+ complexation. The proposed PHBS-sensor can detect Fe3+ ions at ultra-trace levels with limit of detection 1.7 × 10−5 M. Moreover, this applied method was effectively utilized for the monitoring/detection of Fe3+ ions in real water, such as ground, tap and Nile river water, and pharmaceutical samples, such as hard gelatin capsule and syrup.