Background: Identification of cholangiocarcinoma (CCA) associated with hepatolithiasis (HL) is difficult. There is no effective method to discriminate CCA associated with HL (HL-CCA) from HL currently. Objective: To explore the value of clinical data, ultrasonic characteristics and miRNA expression level in the identification of HL-CCA. Methods: Thirty-one patients with HL-CCA in Huazhong University of Science and Technology Union Shenzhen Hospital were enrolled in the observation group, while 40 patients with HL alone were included in the control group. The clinical data, ultrasonic characteristics, and miRNA expression level of the two groups were recorded and analyzed to explore the potential indicators for the identification of HL-CCA. Results: The accuracy of ultrasound in the diagnosis of HL-CCA was low (54.84%). Multivariate logistic regression analysis showed that liver abscess (P=0.021), indistinct border demarcation (P=0.015), non-homogenous echotexture (P=0.019), missed portal vein around lesion (P=0.032), miRNA-21 (P=0.018) and miRNA-221 (P=0.009) were the potential indicators for the identification of HL-CCA. The combined diagnosis based on logistic regression contained liver abscess, border demarcation, echotexture, portal vein around lesion, miRNA-21 and miRNA-221. The results showed that the accuracy of combined diagnosis identifying HL-CCA was the most accurate (AUC=0.911), which was significantly greater than the AUC of miRNA-21 or miRNA-221 individually (P<0.05), with a sensitivity and specificity of 77.42% and 97.50%, respectively. Conclusion: Patients with HL-CCA show high incidence of hepatic abscess and elevated miRNA-21 and miRNA-221 expression level. The ultrasonic features are more likely to show indistinct border demarcation, non-homogenous echotexture, and missed portal vein around lesion. The combined diagnosis is more accurate in the identification of HL-CCA.