We reported a new quinoline-based fluorescent probe 1 for Zn2+ and S2−. 1 showed a highly selective fluorescence enhancement toward Zn2+ over other metal ions including Cd2+, and the resulting 1-Zn2+ complex could detect only S2− among the various anions. The detection limits (0.09 μM and 0.88 μM) for Zn2+ and S2− were much lower than recommended values (76 μM for Zn2+ and 14.8 μM for S2−) by WHO. Their recognition mechanisms were explained by Job plots, UV–vis titrations, 1H NMR titrations, ESI-mass, and theoretical calculations. Moreover, compound 1 with the great photostability and low biotoxicity showed sequentially off-on-off fluorescent bio-imaging of Zn2+ and S2− in both HeLa cells and live zebrafish larvae.