Background & Aims: Late diagnosis is a critical factor undermining clinical management of patients with biliary tract cancer (BTC). While biliary tumours display extensive inter-patient heterogeneity, the host immune response may be comparatively homogenous, providing diagnostic opportunities. Herein, we investigated whether cancer-associated systemic reprogramming could be detected non-invasively to improve diagnosis of BTC. Methods: In this prospective Danish study, whole blood (WB) microRNA (miRNA) profiling was performed in samples from 218 patients with BTC, 99 healthy participants, and 69 patients with differential diagnoses split into discovery (small RNA-sequencing) and validation (RT-qPCR) cohorts. miRNA expression and activity were further investigated in 119 and 660 BTC tissues, respectively. Results: Four WB miRNAs (let-7a-3p, miR-92b-5p, miR-145-3p, miR-582-3p) were identified and validated as diagnostic of BTC on univariable analysis. Two diagnostic miRNA indexes were subsequently identified that were elevated in patients with BTC and in patients with differential diagnoses, compared to healthy participants. The combination of these miRNA indexes with serum CA 19-9 significantly improved the diagnostic performance of CA 19-9 alone, consistently achieving superior AUC values irrespective of clinical setting (minimum AUC >0.84) or tumour location (minimum AUC >0.87). The diagnostic information captured by miRNA indexes was not recapitulated by routine clinical measurements. Index miRNA expression in BTC tissues was associated with distinct pathobiological and immune features. Conclusions: WB miRNA profiles are altered in patients with BTC. Quantification of miRNA indexes in combination with serum CA 19-9 has the potential to improve early diagnosis of BTC, pending further validation. Lay summary: Surgery is currently the only curative intervention for patients with biliary tract cancer (BTC). However, resection is not possible for most patients who are diagnosed with late-stage disease. With the aim of identifying new early diagnostic opportunities, we analysed circulating microRNAs (small non-coding RNAs whose role in cancer is being increasingly recognised) in whole blood samples. We identified a microRNA signature that could distinguish patients with BTC from healthy participants. These miRNAs significantly improved the diagnostic potential of the routinely measured biomarker, CA 19-9, and were implicated in distinct immune processes in tumour tissues.