A new dual-responsive chiral cystine based chemosensor, Cys(cou)2, has been designed and characterized by 1H NMR, 13C NMR, FT-IR, UV-vis as well as elemental analysis. This sensor exhibited an excellent response towards Fe3+ and CN- with high selectivity and sensitivity by fluorescence turn-off mechanism. The binding mode of Cys(cou)2 with Fe3+, and CN- was confirmed by ESI-MS, 1H NMR, and fluorescence titration and also quantum chemical calculation. These results showed that the stoichiometric ratio of Cys(cou)2-Fe3+ and Cys(cou)2-CN is 1:1 and 1:3 in DMSO/Tris aqueous buffer (1:1, v/v), respectively. The linear relationship of the Stern-Volmer plot illustrates the static quenching mechanism at different concentrations. The detection limit (LOD) and binding constant (Ka) for Fe3+ and CN- are 0.029 μM, 1.28 × 104 and 0.51 μM, 9.94 × 106, respectively. Moreover, Cys(cou)2 can act as a colorimetric sensor for CN- in DMSO with the color change from colorless to yellow.