Background: Bronchial washing fluid (BWF) is a less-invasive specimen. Due to the limited sensitivity of BWF cellular component diagnosis, the aim of this study was to explore the potential role of BWF supernatant as a source of liquid biopsy of lung cancer. Methods: This prospective study enrolled 76 suspected and 5 progressed lung cancer patients. Transbronchial biopsy tissues, BWF supernatant (BWF_Sup) and BWF precipitant (BWF_Pre) were tested by a targeted panel of 1021 genes. Results: BWF_Sup cell-free DNA (cfDNA) was superior to tissue biopsy and BWF_Pre in determining mutational allele frequency, tumour mutational burden, and chromosomal instability. Moreover, BWF_Sup and BWF_Pre achieved comparable efficacy to tissue samples in differentiating malignant and benign patients, but only BWF_Sup persisted differentiated performance after excluding 55 malignancies pathologically diagnosed by bronchoscopic biopsy. Among 67 malignant patients, 82.1% and 71.6% of tumour-derived mutations (TDMs) were detected in BWF_Sup and BWF_Pre, respectively, and the detectability of TDMs in BWF_Sup was independent of the cytological examination of BWF. BWF_Sup outperformed BWF_Pre in providing more subclonal information and thus might yield advantage in tracking drug-resistant markers. Conclusions: BWF_Sup cfDNA is a reliable medium for lung cancer diagnosis and genomic profiles and may provide important information for subsequent therapeutic regimens. [ABSTRACT FROM AUTHOR]