Abstract: Background　Breast cancer is characterized by high incidence and marked heterogeneity, which highlights the need for personalized therapeutic strategies. Patient-derived organoids (PDOs) provide an advantageous platform for drug sensitivity testing, but conventional culture systems may be limited by insufficient nutrient supply and other microenvironmental constraints. Microfluidic chip technology can mimic a blood flow-like environment through continuous perfusion and may improve the physiological relevance of PDO culture. Objective　This study aimed to culture breast cancer PDOs using a 3D-printed microfluidic chip and to evaluate the effects of this culture system on PDO growth status, morphological fidelity, and consistency of drug responses, thereby exploring its feasibility for breast cancer PDO culture and individualized drug sensitivity testing. Methods　 Tumor specimens from three invasive breast cancer patients were used to establish PDO. PDOs were cultured under conventional plate conditions and in the microfluidic chip with continuous media perfusion. Hematoxylin & eosin staining and immunohistochemistry for ER, PR, and Ki-67 were performed to compare organoid histology and phenotype with the original tumors. Docetaxel and epirubicin were tested at multiple concentrations to generate dose–response curves and calculate IC50 values. Chip-cultured PDOs were then treated at the corresponding IC50 concentrations to compare drug response consistency between the two culture conditions. Results　Organoids grown in the microfluidic chip were larger and more densely structured, while their H&E histology and marker expression patterns were generally consistent with those from plate culture. Under exposure to the same nominal drug concentration (IC50), there was no statistically significant difference in PDO cell viability between the two culture conditions (P＞0.05), suggesting that the microfluidic culture did not markedly alter PDO drug responses under the present experimental conditions. Conclusion　The 3D-printed microfluidic chip provides a dynamic culture environment that improves PDO growth, and the present findings preliminarily suggest that it does not markedly alter the main phenotypic features or drug response characteristics of PDOs under the tested conditions. This platform may serve as a feasible tool for personalized chemotherapy screening in breast cancer.