A novel di-coordinating rhodamine-based chemosensor, HL with NO donor atoms, selectively and rapidly recognizes Fe³⁺ in the presence of all biologically relevant as well as toxic metal ions and numerous anions and also with other reactive oxygen and nitrogen species. It exhibits a lower detection limit (0.17 μM) and comparatively higher formation constant (K_f = 1.72 × 10⁴ M⁻¹). The DNA-binding properties of [LFe(NO₃)₂]⁺ complex have been comprehensively studied by using UV-Vis, fluorescence, and optical melting studies and circular dichroism, which clearly indicate that [LFe(NO₃)₂]⁺ interacts with DNA via a groove binding mode. In particular, competition experiments with Hoechst and DAPI constitute firm evidence for this binding mode, and clearly rule out intercalation. The negative ΔG⁰ and positive ΔS⁰ values obtained from a calorimetric technique confirm the spontaneity of the binding of [LFe(NO₃)₂]⁺ with DNA. The resulting [LFe(NO₃)₂]⁺/DNA composite material could be a valuable candidate for future photonics and/or biological applications.