Patient-derived cancer organoids emerged as an innovative model in basic and translational medicine research, as well as precision medicine. However, most radiation oncologists are still unaware of the value of organoids in radiation oncology research, especially in precision radiation oncology. The methods for assessment of organoids cell death after irradiation was also undefined. In this study, three organoid lines were successfully established from the surgical specimens of rectal cancer patients. These organoid lines displayed diverse architecture and captured heterogeneity of the tumors. Genetic characterization of rectal cancer organoids showed that the most frequently mutated genes and pathways among rectal cancer tumors also presented in rectal cancer organoids. Our results also revealed that cancer organoids were easy to use for the research of radiobiology of cancer. The survival of rectal cancer organoids after X rays irradiation could be validated by image-based analysis, while the cell viability tested by cell counting kit-8 failed. The survival curves generated from imaging analysis were more representative, which had an initial linear slope, followed by a shoulder, and tended to become straight again. As far as we know, this was the first study to investigate the appropriate testing method of using organoids to study cancer radiosensitivity, suggesting that image-based analysis is more accurate than cell viability.