Distinct “off–on” rhodamine B based chemosensors 3',6'-bis(diethylamino)-2-(2-(diethylamino)ethyl) spiro[isoindoline-1,9′-xanthen]-3-one (2a); 3′,6′-bis(diethylamino)-2-(2-(dimethylamino)ethyl) spiro[isoindole-1,9′-xanthen]- 3-one (2b) are synthesized in a facile single-step reaction and characterized by X-ray crystallography, NMR, Mass, and FT-IR techniques. 2a, 2b; selectively detected Fe+3, Sn+2, and Cr+3, in aqueous media over 17 other environmentally relevant cations. Furthermore, 2a, 2b showed high selectivity towards Fe+3 ion with ∼ 62 fold (2a), ∼21 fold (2b) enhanced emission intensity. A prominent pink color, in < 1 min (∼40 s) was detected under naked eye on sensing Fe+3. Enhanced emission was rationalized by natural transition orbital analysis using DFT and TD-DFT methods. Detection limit and binding constant of Fe+3 for 2a and 2b are estimated as 51.25 ppb (73 nM), 40.3 ppb (59.7 nM), and 0.80 × 103 M−1, 4.59 × 103 M−1, each respectively, indicating their efficiency towards Fe+3 sensing. A test strip method was developed for the on-site qualitative detection of target metal ions, and practical applicability was demonstrated by determining the concentration of Fe+3 ion in real water samples. Probes 2a, 2b further tested in human gastric adenocarcinoma (AGS) cell-lines via in-vitro imaging. The increased emission intensity in 2a, 2b on binding to Fe+3 in AGS cells indicate their potentiality as small-molecule based biological fluorescent probes.