Background: Cholangiocarcinomas (CCAs), which derive from the epithelial cells of the biliary tree, constitute a serious and growing health problem worldwide. Gut and bile microbiota have not been characterized in patients with CCA, and better noninvasive diagnostic approaches for CCA are essential to be established. CA19-9 was the most commonly used tumor marker for CCA; nevertheless, the forecasting performance of CA19-9 remains unsatisfactory. The aim of this study was to investigate the characteristics of gut and bile microbiota combined with CA19-9 in CCA patients. Methods: Fecal samples were collected from 42 CCA patients (16 of the total with Bile samples) and 16 healthy normal controls (HNC). 42 CCA patients were divided into three group according to CA19-9 levels: CCN (CA19-9 negative),CCP (CA19-9 positive: CPL (CA19-9 positive within 10 times) and CPH (CA19-9 positive over 10 times)) groups. DNA was extracted from fecal and bile samples and subjected to 16S rRNA gene analysis. The correlation between gut microbiota and clinical data in CCA group were further analyzed with Spearman rank correlation analysis. Results: Comparing with HNC group, the relative abundance of Firmicutes and Actinobacteriota were significantly decreased, whereas Proteobacteria and Bacteroidota were significantly enriched in CCA group at phylum level (all P < 0.01). Interestingly, Firmicutes/Bacteroidota (F/B) ratio significant decreased in CCA group compared to HNC group(P < 0.05). CA19-9 were inversely correlated with Bifidobacterium, which in turn was associated with Klebsiella in CCA patients. Especially the relative abundance of Klebsiella in CCP group was significantly enriched than CCN group(P < 0.05). Conclusions: Our findings provide evidence supporting Klebsiella may serve as a non-invasive intestinal microbiomarker for CCAs. Combinating with CA19-9, Klebsiella may improve the diagnosis of CCAs as non-invasive approach. Bifidobacterium is likely to be a potential protective factor for anti-tumorgenesis of CCAs.