In this work, two novel fluorescence probes, named as RBQ1 and RBQ2, were designed and synthesized based on rhodamine B lactam. RBQ1 was employed for sensitive fluorometric sensing of H+ in the pH range of 2–4. Since RBQ1 exhibits color responses toward Cu2+, it was also employed for sensitive detection of Cu2+ by photoelectric colorimetry. By using RBQ1 as the fluorescence probe, the detection limit of Cu2+ obtained was 0.0365 μmol L−1. RBQ2, with a similar chemical structure to RBQ1, also shows obvious fluorescence responses to H+, but no response to any metal ions. By using H+ and Cu2+ ions as chemical inputs and fluorescence or absorbance as outputs, a novel dual-mode molecular logic gate was constructed with RBQ1.