Objective In this study, we present a living biobank of patient-derived tumoroids from advanced colorectal cancer (CRC) patients and show examples of how these tumoroids can be used to simulate cancer behavior ex vivo and provide more evidence for tumoroids could be utilized as a predictive platform during chemotherapy to identify the chemotherapy response. Methods The tumor tissues of CRC patients were collected to isolate and culture tumoroids, and the histomorphology of tumoroids was evaluated. Further, tumoroids were treated with drugs of different chemotherapy schemes, and the drug sensitivity of tumoroids was evaluated by using CellTiter-GIo 3D cell viability assay, and the clinical efficacy was compared with that of patients. Results The tumoroids were still highly consistent with the original tumor histomorphology after continuous passage. The consistency between the drug sensitivity of tumoroids from different patients and the clinical efficacy of corresponding CRC patients was 91.18% (31/34). The drug inhibition rate of tumoroids was positively correlated with the progression free survival (PFS) of CRC patients (rs=0.412, P=0.016), while the area under the cell activity drug concentration curve of tumoroids was negatively correlated with the PFS of CRC patients (rs=–0.479, P=0.004). Conclusion This study established a biological sample bank of tumoroids for CRC patients, and suggested that tumoroids had the potential to be used as preclinical experimental models and predict the chemotherapy effect of CRC patients.