Current cancer screening tests, such as low dose computed tomography (LDCT) for lung cancer and colonoscopy for colorectal cancer, are challenged by low sensitivity, high false positive rate, limited tumor information, uncomfortable or invasive procedures and high cost. Liquid biopsy that detects circulating biomarkers, allows sequential monitoring of cancer development, complements medical imaging and risk factor data to provide a more comprehensive portrait of cancer, and thus it has shown great promise as the in vitro diagnostics for cancer. Exosomal proteins and exosomal microRNAs are promising cancer biomarkers for liquid biopsy, and have shown very high sensitivity and specificity in diagnosing many cancers. Unfortunately current detection technologies, such as ELISA, LC-MS, qRT-PCR, microarray and next generation sequencing, are tedious, expensive and time consuming, which limits their clinical utilities as screening tests. Moreover, many methods cannot distinguish tumor-derived exosomes from their non-tumor counterparts, and thus suffer from poor detection sensitivity and specificity. To address these challenges, we have developed a surface plasmon resonance (SPR)-based, molecular beacon-assisted liquid biopsy device, named Exosome Protein microRNA OneStop (Exo-PROS) biosensor. This biosensor can capture tumor-derived exosomes using cancer-overexpressed proteins. It can also provide one-stop quantification of surface proteins and intravesicular microRNAs of captured exosomes on a single device within 4 hours. The effective separation of tumor-derived-exosomes from interfering exosomes (such as normal-cell-derived exosomes) and the combination of exosomal protein and microRNA biomarkers may significantly increase the diagnostic accuracy. We have successfully used the Exo-PROS biosensor to measure exosomal EGFR and EGFR+ exosomal miR21 in cell culture medium and lung cancer patient serum samples. The biosensor showed higher sensitivity and specificity than ELISA and qRT-PCR in distinguishing cancer from normal controls. In this study, we propose to (1) advance the development of the Exo-PROS biosensor to improve its sensing performance and to realize multiplexed biomarker detection; (2) demonstrate its clinical utility in lung cancer screening and early detection, and explore the diagnostic value of exosomal protein and microRNA combined biomarkers in cancer, which has not yet been investigated. We will evaluate the detection sensitivity and specificity of Exo-PROS biosensor in distinguishing nonsmall cell lung cancer patients from healthy controls and smokers with benign solitary pulmonary nodule at high risk for lung cancer. We will demonstrate the superior sensing performance of ExoPROS biosensor over ELISA and qRT-PCR assays. We expect to develop the Exo-PROS biosensor into a highly accurate, simple, fast, and cost-effective liquid biopsy test for cancer screening and early detection.