Current methods for the early diagnosis of cancer can be invasive and costly. In recent years, exosomes have been recognized as potential biomarkers for cancer diagnostics. The common methods for quantitative detection of exosomes, such as nanoparticle tracking analysis (NTA) and flow cytometry, rely on large-scale instruments and complex operation, with results not specific for cancer. Herein, we present a tri-channel electrochemical immunobiosensor for enzyme-free and label-free detecting carcino-embryonic antigen (CEA), neuron-specific enolase (NSE), and cytokeratin 19 fragments (Cyfra21-1) from exosomes for specific early diagnosis of lung cancer. The electrochemical immunobiosensor showed good selectivity and stability. Under optimum experimental conditions, the linear ranges were from 10⁻³ to 10 ng/mL for CEA, 10⁻⁴ to 10² ng/mL for NSE, and 10⁻³ to 10² ng/mL for Cyfra21-1, and a detection limit down to 10⁻⁴ ng/mL was achieved. Furthermore, we performed exosome analysis in three kinds of lung cancer. The results showed a distinct expression level of exosomal markers in different types. These works provide insight into a promising alternative for the quantification of exosomal markers in specific diseases in the following clinical bioassays.