A new quinoline based chromo − fluorogenic chemosensor (QMEH) was designed for the sequential recognition of Cu2+ and N–acetyl–L–cysteine (NAC) in DMSO:H2O (50:50, v/v) medium. Excellent visual color changing and fluorescence quenching of QMEH with gradually adding of Cu2+ were monitored with highly selective binding stoichiometry (1:1), which might be resulted from a chelation enhanced quenching (CHEQ) phenomenon. Next, the Cu2+−enhanced absorbance and − quenched emission of QMEH were fully recovered by NAC through the displacement approach. The QMEH−Cu2+ complex structures showed obvious color changes by the adding of NAC from ’’bright yellow’’ to ’’noncolored’’ under daylight and from ’’noncolored’’ to ’’blue’’ under UV−cabinet, respectively. The gradual addition of NAC to the QMEH−Cu2+ solution induced its absorbance and emission recovery with low detection limits of 65.88 μM and 6.32 μM, respectively. To assess the importance of method parameters, the validation study was carried out using diverse analytical parameters. Moreover, thanks to the superior sensing performance and the low cytotoxicity ability of QMEH, it was used for the Smartphone platform, natural spring waters and the sequential bioimaging of Cu2+ and NAC in hepatocellular carcinoma cells.