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Exosomes are closely associated with tumor development and are regarded as viable biomarkers for cancer. Here, a ratiometric fluorescence method was proposed for the one-step and label-free detection of plasma exosomes. A bicolor streptavidin magnetic beads were specifically created with an immobilized Cy5-labeled hairpin aptamer for CD63 (Cy5-Apt) on its surface to identify exosome, and a green color SYBR Green I (SGI) embedded in the stem of Cy5-Apt to respond to exosomes. After exosome capture, the Cy5-Apt could undergo a conformational shift and release the encapsulated SGI, allowing exosome measurement based on the fluorescence ratio of Cy5 and SGI. The enrichment, separation and detection of exosomes in proposed method could be completed in one step (30 min), which is a significant improvement over previous method. Furthermore, the use of ratiometric fluorescence and magnetic separation allows for exosome enrichment and interference elimination from complex matrices, improving accuracy and sensitivity. Particularly, the assay could detect exosomes in plasma and has potential to distinguish lung cancer patients from healthy volunteers with an area under the receiver operator characteristic curve of 0.85. Besides, the study provided an efficient method for analyzing the various divisions of exosomes by merely modifying the aptamer, which holds great promise for point-of-care applications. A one-step , label-free ratiometric fluorescence assay was developed for the accurate and point-of-care detection of exosome from plasma and its application in cancer diagnosis. [Display omitted] • The proposed method can achieve the integration of exosome separation and detection. • The self-calibration of MNPs-Apt@SGI can eradicate environmental effects and enhance sensitivity. • The assay enables one-step and label-free analysis within 30 min, which is great shorter than other methods. • The AUC of the established method in cancer diagnosis achieved a value of 0.85. • The strategy can extend to sub-population exosomes analysis by merely modifying aptamer. [ABSTRACT FROM AUTHOR]