© 2022 Elsevier B.V.A new dual-channel probe based on rhodamine B derivative (MSB) was successfully designed, synthesized, characterized by Nuclear Magnetic Resonance (NMR) Spectroscopy, Fourier Transform Infrared Spectrophotometer (FTIR), Matrix Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry (MALDI-TOF MS), X-ray Photoelectron Spectroscopy (XPS), and Single Crystal X-ray Diffraction, and the sensing abilities toward Fe3+ cation have been demonstrated and the probe was successfully utilized for fluorescence imaging of Fe3+ in living cells. The probe demonstrated quite fast, sensitive, and selective response to Fe3+ by causing an extreme enhancement in UV–vis and fluorescence spectroscopy techniques in the buffered aqueous media which makes MSB a dual-channel probe. While the color of MSB solution was initially light yellow, it turned pink in the presence of Fe3+, which provided highly selective naked-eye determination among several ions as alkaline, alkaline-earth, and transition metal ions. After that, the probe was easily applied to paper strips and real samples such as drinking waters and supplementary iron tablets for sensing Fe3+ in an aqueous solution. The detection limit (LOD) and the response time of the probe were determined as 4.85x10-9 M and 4 min, respectively, which are quite lower compared with other rhodamine based Fe3+ sensors in the literature. According to Job's plot, 1H NMR titration, MALDI-TOF MS, XPS, and DFT study techniques, the complexation ratio between MSB and Fe3+ was found as 1:1. Moreover, the spectral response was reversible with alternately addition of Fe3+ or Na2EDTA to the MSB solution. In addition, fluorescence imaging in NIH/3T3 mouse fibroblast cells and studies in real samples with a quite high recovery rate exhibited that the probe is qualified for detection of Fe3+ ion with multiple practical usages.