In this work, a dual-functional Cu2+-based ensemble (2S·Cu2+) was well designed and characterized. Then, the successional and discriminating sensing for CN− over other competitive species (H2PO4− and biothiols) was achieved based on the disaggregation of 2S·Cu2+ ensemble and the deprotonation of imidazole NH of regenerated sensor S in aqueous solution, respectively. The visual sensing mechanism could be clearly demonstrated by 1H NMR, HRMS and energy changes between the HOMO−LUMO band gaps. Furthermore, the reversibility and reusability of S and 2S·Cu2+ upon alternating addition of CN−/H+ and CN−/Cu2+ were studied. Interestingly, the sequential sensing for biothiols (cysteine, glutathione and homocysteine) and CN− was also realized through spectroscopic methodology and test paper strips. This work may provide a feasible strategy to discriminate CN− over H2PO4− and biothiols with high selectivity and sensitivity through Cu2+-based ensembles.