<p>Total quantification and speciation of Fe²⁺ and Fe³⁺ at trace levels in aqueous environments has emerged as an imperative analytical challenge for ecological systems and public health. Implementation of wavelength-dependent spectrochemical methodologies based on formation of chromogenic Fe³⁺ chelate for Fe³⁺ quantification in aqueous media also remains intrinsically challenging owing to intrinsic self-absorbance, and spectral convolution. Thus, the current study reports the development of a direct and selective eco-friendly spectrofluorometric probe for Fe³⁺ detection in water. The sensor 4, 5- dihydroxy-1, 3-benzenedisulfonic acid (Tiron) displays strong emission which undergoes significant quenching in the fluorescence intensity (at λ_ex/em = 290/350 nm) upon reaction with Fe²⁺ at pH 8–9 in the presence of interfering metal ions, allowing for rapid and efficient detection. The probe was sensitive to implement the produced change in color with a total assay reaction time less than 3&#xa0;min. Detailed spectroscopic investigation reveal a high binding affinity between the Fe³⁺ ions and the sensor, ascribed to specific complex formation. The limit of detection (LOD) and quantitation (LOQ) and linear dynamic rang (LDR) of the assay were 0.015, 0.04 and 0.05-5 mg L⁻¹ Fe³⁺, respectively. The probe was favorably used for total detection and sequential speciation of trace levels of Fe²⁺ &amp; Fe³⁺ in water after oxidation of the former to Fe³⁺. Spectroscopic studies suggested high coordinating attraction and stability between the sensor and Fe³⁺ ions. The probe was fruitfully validated by comparing the results with the official inductively coupled plasma–optical emission spectrometry (ICP-OES) data using <i>Student t and F</i> tests signifying the high precision, accuracy and reliability for Fe³⁺ detection in water samples. The attractive features of the probe comprises its simplicity, no usage of organic solvents, short reaction time; high reactivity, stable response, selective towards Fe³⁺, and sensitive for total quantification of iron species. The probe can assist as a talented tool for application in regions of limiting resources specifically concerning iron pollution.</p>