Abstract Tumor‐derived exosomes are crucial for early non‐invasive and accurate tumor diagnosis in clinical diagnostics. The development of highly sensitive, simple, and intuitive exosome assays has sparked a research upsurge in clinical diagnostics. Here, we develop a bio‐responsive intelligent DNA hydrogel loaded with CRISPR/Cas12a for universal and ultrasensitive detection of the exosomes. The aptamer serves as the target response unit and switch, competitively disintegrating the region of the DNA linkers and then Cas12a/crRNA was released and activated, resulting in a high fluorescent intensity for exosome detection at the detection limit of 119 particles/μL. Moreover, a constructed colorimetric tube is made by loading a colorimetric filter membrane on the tube lid and intelligent DNA hydrogel on the tube bottom, which enables one‐pot portable colorimetric detection. Without the need for laboratory instruments and professionals, this strategy allows for naked eye detection with limit of detection as low as 104 particles/μL, and shows great applicability in distinguishing between healthy individuals, pretreatment patients, and post‐treatment patients after obtaining a testable analyte. In this study, an ultrasensitive detection platform for exosomes that enables one‐step sensing and dual signal output was introduced. The findings here suggest that this platform is a promising tool for the application of liquid biopsy based on exosomes in clinical diagnosis.
