We first characterized the physical and biological properties of lung cancer cell line (i.e., HCC827, H1975, and HCC78) derived sEVs, including morphology, size distribution, and expression of lung‐cancer‐associated biomarkers (i.e., THBS2, VCAN, and TNC) and canonical tetraspanin (i.e., CD9, CD63, and CD81) to establish and study the validity of cell line sEVs for lung cancer screening (Figure 2). Here, VCAN is linked to lung cancer.