To investigate the antitumor properties of copper(Ⅱ) complexes, a series of Cu(Ⅱ) complexes (C1-C3) derived from 6, 7-dihydro-5H-quinoline-8-one thiosemicarbazone ligands was designed and synthesized. These complexes exhibited significantly higher potency in inhibiting tumor cell growth in vitro compared to cisplatin. Among them, C3 had the highest antitumor activity against MDA-MB-231 cells, with a half maximal inhibitory concentration (IC₅₀) value of 1.42 μmol·L^-1. Moreover, C3 effectively inhibited the growth of 3D multicellular spheres. Mechanistically, it induced significant reactive oxygen species (ROS) generation, initiating a dual-pathway cytotoxic effect. On the one hand, it triggers endoplasmic reticulum stress and inhibits the activity of the related protein, protein disulfide isomerase (PDI). On the other hand, it induces mitochondrial dysfunction. These combined stresses ultimately lead to the apoptosis of MDA-MB-231 cells.