A novel rhodamine-based colorimetric and fluorescent sensor for Hg2+ was reported. In CH3CN/H2O (1/99, v/v), the sensor showed high selectivity and sensitivity to Hg2+ by an emerging absorption peak at 565 nm and a 32-fold fluorescence enhancement at 586 nm with vivid color changes. The absorbance and fluorescence maxima increased linearly with the concentration of Hg2+ in the range of 0–90 μM. The colorimetric and fluorescent detection limits were 6.36 μM and 60.78 nM respectively. The sensor could work in a nearly neutral pH span of 6.01–8.57 and exhibited excellent interference immunity and low cytotoxicity. A 1:2 sensor-Hg2+ complex formed with a binding constant of 2.89 × 108 M−2. A new sensing mechanism was proposed. The sensor was successfully applied in real sample assay and living cell imaging. Furthermore, the sensor could be supported in low cost cellulose discs for signaling Hg2+ in 100% aqueous solution.