ObjectiveBiliary tract cancer (BTC) is a rare malignancy and lack of effective diagnostic and prognostic marker. Here, we aimed to investigate the clinical implication of TP53 mutation detection in BTC using droplet digital PCR (ddPCR).MethodsTP53 gene (loci p.R175H, p.R248Q, p.R248W, and p.R273H) mutation frequencies of 45 pairs of tumor tissues (TTs) and adjacent normal tissues (ANTTs) were analyzed, respectively, using ddPCR. Meanwhile, the same detections were conducted in plasma cell‐free DNA (cfNDA) of 156 subjects including BTC, disease control (DC), and healthy controls (HC). The logistic regression algorithm was established to identify BTC. The correlations between mutations and clinicopathological features as well as the effects of TP53 mutation frequency on BTC prognosis were assessed.ResultsThe higher mutation of p.R175H was found in TTs compared with ANTT (p = 0.006). The mutation at p.R273H in cfDNA was also higher in BTC when compared with DC and HC (p < 0.05). The logistic algorithms combining p.R273H mutation demonstrated the higher diagnostic efficacy trend than carbohydrate antigen 19–9 (CA19‐9), carcinoembryonic antigen (CEA), and alpha‐fetoprotein (AFP) in identifying BTC from DC (the area under the curves of the algorithm: 0.845, 95% CI:0.775–0.914). The median overall survival (OS) and progression‐free survival (PFS) were significantly shorter when the BTC patients harboring the p.R273H mutation (OS: p = 0.032; PFS: p = 0.046).ConclusionThis study revealed for the first time that the quantitative TP53 mutations using the ddPCR might serve as a potential genetic biomarker for BTC diagnosis and prognosis assessment.