<p>Iron(III) and copper(II) ions are ubiquitous in the environment, posing potential risks to the environment and human health, which urgently demands rapid detection methods. In this study, a ratiometric fluorescent probe was developed by integrating biomass-based carbon dots with rhodamine B (RhB) for the rapid and accurate detection of Fe³⁺ and Cu²⁺. Under normal conditions, the strong coordination ability of Fe³⁺ and Cu²⁺ enables them to rapidly occupy the active sites on the surface of carbon dots (CDs), resulting in rapid fluorescence changes. RhB provides a stable reference signal and achieves self-calibrated dual-emission output, thereby reducing matrix and environmental interferences. To address the coexistence of Fe³⁺ and Cu²⁺ in practical detection, ethylenediaminetetraacetic acid (EDTA) was introduced as a selective masking agent, which preferentially forms a stable complex with Fe³⁺. This reduces the signal interference of Fe³⁺ in low-concentration systems and realizes highly selective detection of Cu²⁺. In the detection of Cu²⁺ in actual water samples, the probe exhibits high sensitivity (4.91 µM) and favorable accuracy with recoveries ranging from 97.3% to 99.5%, demonstrating strong reliability in complex samples. This study not only provides a green and sensitive method for Cu²⁺ detection, but also presents an effective strategy for resolving specific detection issues in complex matrices using classical masking agents.</p>