Exosomes are emerging as one of the most promising biomarkers for early disease diagnosis and prognosis. The significant challenges facing the available methods include improving the detection specificity and sensitivity in complex biological samples. Herein, a fluorescence assay was established based on a combination of immunomagnetic separation and a two-step signal amplification strategy for direct isolation and subsequent detection of exosomes. First, immunomagnetic beads capture and enrich the exosomes via antibody-antigen reactions. Second, bivalent cholesterol (BC) anchors spontaneously insert into the lipid bilayer of bead-captured exosomes, forming a "one to many" amplification effect. The simultaneous recognition of the surface protein and the lipid bilayer structure of the exosome significantly eliminates the interference risk from free proteins. The detection of exosomes converts to the detection of BC-anchors. Finally, the sticky end of the BC-anchor acts as the initiator to trigger the enzyme-free DNA circuits for secondary signal amplification. Under the optimal conditions, highly sensitive and selective detection of exosomes was achieved ranging from 5.5 × 10 to 1.1 × 10 particles/μL with a limit of detection of 1.29 × 10 particles/μL. Moreover, this method allows the isolation and quantitative analysis of exosomes in several biological fluids with satisfactory recovery rates (92.25-106.8%). Thus, this approach provides a sensitive, anti-interference platform for isolating and detecting exosomes.