This multicenter retrospective study investigated the clinical utility of patient-derived tumor organoids (PDOs) in guiding adjuvant chemotherapy for intrahepatic cholangiocarcinoma (ICC) patients. A total of 82 ICC patients at the Shanghai Eastern Hepatobiliary Surgery Hospital and the Shanghai Tenth People’s Hospital between 2021 and 2022 were included in the study. All of these patients underwent surgical resection and were pathologically confirmed to have high-risk factors for recurrence. Therefore, they received adjuvant chemotherapy post-surgery. Sixty-one PDOs were successfully established from resected tumor tissues and tested against four common regimens: Gemcitabine + cisplatin, gemcitabine + oxaliplatin, capecitabine, and S-1. A novel fluorescence-based method was developed for drug sensitivity testing, showing significant concordance with conventional half-maximal inhibitory concentration quantification. Patients were stratified into matched (n=26) and unmatched (n=35) groups based on alignment between PDOs-based drug sensitivity results and actual chemotherapy received. The matched group demonstrated significantly improved recurrence-free survival (RFS) (hazard ratio = 2.52, p=0.007), with 63% versus 17% experiencing recurrence events. Through eXtreme Gradient Boosting machine learning and Cox regression analyses, we identified PDOs-based drug sensitivity, tumor multiplicity, and vascular invasion as key predictors of 2-year recurrence. A clinical nomogram incorporating these factors showed that patients with matched PDO-guided therapy had 46% 12-month RFS probability versus 28% in unmatched cases. This study provides a clinically translatable platform to guide adjuvant chemotherapy decisions and predict the prognosis of ICC patients with a high recurrence risk after surgery.