Intrahepatic cholangiocarcinoma (ICC) is a highly lethal malignancy associated with significant morbidity, necessitating the urgent development of an effective chemotherapeutic assay for ICC patients. In this study, we have successfully established an advanced culture method for ICC organoids that can be utilized with both single-cell assembly and tissue fragmentation initiation techniques. These ICC organoids maintain the morphological characteristics, including mutation profiles and frequency (46.9% in organoid and 48.5% in tumor tissue) of IDH1 genes, and 1733 high-frequent overlapped mutated genes (94.2%). Additionally, ICC biomarkers such as CK7 and CK19 also maintain a similar pattern compared with the original tissue. Furthermore, RNA-seq analysis reveals upregulation of immune-related genes in single-cell assembly organoids. The significantly changed genes including IL9R (4.4-fold), IL2RB (3.2-fold), CCR4 (3.5-fold), TESPA1 (4.4-fold), ZAP70 (4.3-fold) and CD6 (4.3-fold) in log scale. These evidence both indicating the presence of viable and active immune cells. Overall, our findings present an advanced and user-friendly culture approach for generating ICC organoids adaptable to diverse experimental objectives.