OBJECTIVE This study aims to establish and characterize patient-derived colorectal cancer organoid models and evaluate their potential value in predicting chemotherapy responses through individualized drug sensitivity testing. METHODS Tumor tissues were obtained from surgical specimens of three patients who underwent radical surgery for colorectal cancer. Tumor cells were isolated through mechanical and enzymatic dissociation, and the resulting cell suspension was mixed with Matrigel and seeded into a three-dimensional culture system. The histopathological characteristics of the organoids were validated using hematoxylin-eosin (HE) staining and immunohistochemistry, and their genetic consistency was confirmed by short tandem repeat (STR) analysis, demonstrating that this model could reflect both intra- and inter-patient tumor heterogeneity. Standard clinical chemotherapy regimens were applied to the organoid models, and drug sensitivity was assessed using the CellTiter-Glo^® 3D cell viability assay. RESULTS The cultured colorectal cancer organoids closely resembled their originating tumors in terms of histopathological and genetic characteristics. Drug sensitivity testing indicated that organoids derived from different patients exhibited varied sensitivities to commonly used chemotherapeutic agents. CONCLUSION The colorectal cancer organoid model successfully established in this study closely recapitulates the histological classification and genetic features of the parent tumors and shows potential for application in vitro chemotherapy sensitivity testing. This model could be used to predict colorectal cancer patients’ responses to chemotherapy, providing valuable reference for personalized medicine.