A selective chromone‐containing colorimetric chemosensor DOCC ((E)‐N,N‐dimethyl‐2‐((4‐oxo‐4H‐chromen‐3‐yl)methylene)hydr azine‐1‐carbothioamide) was designed for the sequential probing of Cu2+ and cysteine. DOCC exhibited a marked color variation from colorless to pale yellow and had the lowest limit of detection (0.30 μM) among the chromone‐based colorimetric sensors for Cu2+ in near‐perfect aqueous media. DOCC could work to probe and quantify Cu2+ in real water samples with great recovery (98‐102%). DOCC could discriminate Cu2+ using the test kit down to 10 μM. On the other hand, Cu2+‐2⋅DOCC could selectively detect cysteine (Cys) in a sequential step through the demetallation reaction. Cu2+‐2⋅DOCC showed that its color returned from pale yellow to colorless only in the presence of Cys and its limit of detection was calculated to be 6.62 μM. The practical applications for Cys were successfully achieved in water samples with perfect recovery (99–100%) and pH tests at 7–9. FT‐IR, Job plot, ESI‐MS, and DFT calculations were applied to explain the sensing mechanisms of Cu2+ by DOCC and Cys by Cu2+‐2⋅DOCC.