A simple structure, a rhodamine fluorescent derivative, was synthesized easily by a one-step condensation reaction of rhodamine B ethylenediamine and acetylacetone. Its structure was characterized by X-ray crystallography, NMR, MS and IR spectroscopy. As a bi-functional and highly selective “OFF–ON” chemosensor for Fe 3+ or Cu 2+, the derivative displayed a selective fluorescence enhancement effect only with Fe 3+ and an absorption enhancement effect only with Cu 2+ by modulating the pH value. In an aqueous ethanol medium, Fe 3+ formed a 1:1 complex with the derivative at pH 6, while Cu 2+ formed a 1:1 complex at pH 8.5. The limits of detection of the ions were low: 3.9 × 10 −9 and 4.8 × 10 −8 mol L −1. The derivative also functioned as a fluorescence sensor for CT-DNA, in which the Fe 3+ ion that was used as a bridge between the derivative and DNA forming a ternary complex.