Purpose <p>Liquid biopsy technology has received widespread attention in the early diagnosis of cholangiocarcinoma (CCA).</p> Methods <p>We collected bile samples from 48 patients with CCA and 48 patients with gallstones at Shandong Provincial third Hospital. We quantified bile circulating free DNA (cfDNA) of syncytin-1 and SLC7A11, calculated the correlation between syncytin-1 and SLC7A11 expression and clinical parameters by Spearman rank correlation, plotted Receiver Operating Characteristic (ROC) curves, and compared the Area Under Curve (AUC) values to explored early diagnostic utility in patients.</p> Results <p>We first found the bile cfDNA of syncytin-1 and SLC7A11 levels in CCA were higher than in gallstones (3.06 vs. 1.32, <i>p</i> &lt; 0.001; 2.39 vs. 1.30, <i>p</i> &lt; 0.001). And there was significant correlation between syncytin-1 and SLC7A11 expression (<i>p</i> = 0.025). Additionally, bile cfDNA of syncytin-1 or SLC7A11 was differentially expressed in gallstones, cholangiocarcinoma stage I-II, and cholangiocarcinoma stage III-IV (<i>p</i> &lt; 0.001; <i>p</i> &lt; 0.001). The AUC of bile cfDNA of syncytin-1 was 0.805 (<i>p</i> &lt; 0.001, specificity of 87.0%), the AUC of bile cfDNA of SLC7A11 was 0.755 (<i>p</i> &lt; 0.001, specificity of 80.4%), and combination of bile cDNA of syncytin-1/SLC7A11/CA19-9 markers improved diagnostic efficiency in CCA patients (AUC: 0.927, <i>p</i> &lt; 0.001).</p> Conclusion <p>The bile cfDNA of syncytin-1 and SLC7A11 was high expression in cholangiocarcinoma, which may be used as a novel biomarker for early diagnosis.</p>