This study was designed to present a new quercetin encapsulated chitosan functionalized copper oxide nanoparticle (CuO-ChNPs-Q) and assessed its anti-breast cancer activity both in vitro and in vivo. The CuO-ChNPs-Q may act as anti-proliferating agent against DMBA-induced mammary carcinoma in female rats. The CuONPs was functionalized with chitosan then quercetin was conjugated with them producing CuO-ChNPs-Q, then characterized. The in vitro anti-proliferating activity of the CuO-ChNPs-Q was evaluated against three human cell line. Then, the anti-breast cancer effect of the CuO-ChNPs-Q was assessed against DMBA-induction compared to both CuONPs and Q in female rat model. The in vitro results proved the potent anticancer activity of the CuO-ChNPs-Q compared to CuONPs and quercetin. The in vivo data showed significant reduction in breast tumors of DMBA-induced rats treated with CuO-ChNPs-Q compared to CuONPs and Q. The CuO-ChNPs-Q treatment had induced apoptosis via increased p53 gene, arrested the cell-cycle, and increased both cytochrome c and caspase-3 levels leading to mammary carcinoma cell death. Also, the CuO-ChNPs-Q treatment had suppressed the PCNA gene which decreased the proliferation of the mammary carcinoma cells. In conclusion, the CuO-ChNPs-Q might be a promising chemotherapeutic agent for treatment of breast cancer with a minimal toxicity on vital organs.