Based on 1,2-dimethoxyphenyl (veratrole, open) and 1,2-methylenedioxyphenyl (pepper ring, close)-derived pyridine–triazole analogues, two groups of copper(II) complexes, namely, Group I(C1–C3) and Group II(C4–C6) were synthesized and fully characterized. All ligands and complexes were tested in vitro by MTT assays on seven tumour cell lines (T24, Hep-G2, Sk-Ov-3, MGC-803, HeLa, A549 and NCI-H460) and one normal liver cell line (HL-7702). Surprisingly, the pepper-ring-derived complexes (C4–C6) showed significantly enhanced cytotoxicity compared with the 1,2-bimethoxyphenyl ring-derived complexes (C1–C3) and the standard anticancer drug cisplatin. Cellular uptake assays indicated that the Cu accumulation was consistent with cytotoxicity. In addition, flow cytometry and western blot analysis showed that the apoptosis of the leading complex C4 may be induced by the Bcl-2 family-mediated proteins through the mitochondrial dysfunction pathway. Furthermore, UV-vis and fluorescence spectroscopy assays revealed that C4 has stronger insertion-binding interactions with CT-DNA than C1 and the fluorescence of C1 and C4 with BSA is mainly quenched by static quenching.