Abnormally expressed surface proteins on tumor extracellular vesicles (tEVs) are ideal noninvasive biomarkers for cancer diagnosis, whereas isolation-integrated profiling of tEVs in biofluids remains challenging. Herein, we show a bubble multimer-enhancing fluorescence (BMEF)-driven extracellular vesicles (BMEF-EV) assay that streamlines rapid enrichment and sensitive profiling of plasma tEVs for the accurate diagnosis of colorectal cancer (CRC). By assembling antifouling nanofilm, clickable moieties, and antibodies on bubbles in sequence, this assay leverages the multifunctional bubbles to streamline isolation and profiling of tEVs in a single test. In BMEF-EV, the buoyant multifunctional bubbles with captured EVs can spontaneously gather at the gas-liquid interface of the droplet apex, showing a BMEF effect to enable direct and sensitive tEV assay. By profiling multiple CRC EV markers in plasma samples from a pilot cohort, this assay achieves early diagnosis of CRC with a high accuracy of near 90%. This Lab-on-Bubbles technology provides a self-contained tool to enable accurate blood-based cancer diagnosis.