Background: Although many studies have confirmed a relationship between microRNAs (miRNAs) and cholangiocarcinoma (CCA), the real-time dynamics of miRNA function have not been examined. Methods: miRNA reporter constructs were generated using a recombinant adeno-associated virus vector, which contained complementary sequences for six miRNAs (miR-200a, miR-200b, miR-21, miR-146a, miR-155, and miR-221), along with two independent expression cassettes encoding the fluorescent reporter genes Fluc and Gluc. The spatio-temporal function of each miRNA was monitored both in CCA and control tissues. Results: All miRNAs participated in CCA development, with distinct patterns of expression over time. The activity of miR-21 was significantly lower in female T3N0M0 CCA tissue relative to controls at three time points, yet was higher in two male T3N1M0 CCA tissues. The difference in miR-200b function between two male T3N1M0 CCA tissues and their corresponding controls peaked at 24 h, while function in a female T3N0M0 CCA was detected only at 72 h. The four remaining miRNAs (miR-200a, miR-146a, miR-155, and miR-221) displayed patient-specific activity patterns in both CCA and control tissues. Conclusion: Significant variability was observed in the temporal function of all six miRNAs, which may play an important role in the development of CCA.