Cholangiocarcinoma (CCA) is a lethal disease owing to its difficulty to be diagnosed at early stages and lack of effective treatment. To improve the accuracy of early diagnosis of CCA, one of the promising approaches is to use CCA-specific affinity reagents to detect CCA cells by liquid biopsy. In this study, bile juice, a body fluid close to lesions, was used as a clinical sample for CCA cell isolation and detection. Moreover, a novel microfluidic chip was designed and fabricated to detect CCA cells in the bile juice by using three types of affinity reagents. Experimental results showed that the three affinity reagents could capture CCA tumor cells in patients' bile juice (with a volume of only 3 mL) within 120 min. This microfluidic system may serve as a promising tool for detecting CCA in clinical settings.