Esophageal cancer is a major global health challenge, with high incidence and mortality due to the lack of rapid and sensitive diagnostic tools and specific biomarkers. Cancer-cell-derived extracellular vesicles (EVs) carry unique proteins and nucleic acids, making them valuable sources of cancer biomarkers. We report an integrated method that combines an ultrafast exosome isolation system (EXODUS) with matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) to detect EVs and identify protein biomarkers for diagnosing and monitoring esophageal squamous cell carcinoma (ESCC). EVs derived from 20 mL culture medium supernatant of ESCC cells with varying degrees of differentiation serve as analysis models. We use EXODUS to isolate EVs rapidly. We then analyze the intact EVs using MALDI-TOF MS, which provides cell line-specific EV fingerprints in minutes. These protein fingerprints allow the discrimination of ESCC from normal control cells and enable the classification of ESCC based on the degree of cell differentiation. We explore critical EV biomarker peaks for ESCC diagnosis (5555