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Since the discovery of exosomes, their potential diagnostic value has been the focus of considerable research. However, the lack of a rapid and simple technique for the quantitative analysis of exosomes greatly limits the application of exosomes in clinical research. In this study, we describe a newly developed one-step chemiluminescence immunoassay for the rapid quantitative analysis of exosomes from biofluids. Our new technique, named ExoNANO, adopts a double-antibody sandwich strategy using anti-CD63 antibody-conjugated superparamagnetic iron oxide particles (SIOPs) and acridinium ester (ACE)-labeled anti-CD9 antibodies. SIOPs have narrow size distribution and high magnetic susceptibility, and ACE has excellent chemiluminescent properties such as low background signal and no need for a catalyst. We demonstrated that ExoNANO allows the quantitative analysis of exosomes in the range of 2.92 ×10 5 to 2.80×10 8 particles/ μ L, with a limit of detection of 2.63×10 5 particles/ μ L. Using ExoNANO, we quantified exosomes in cell culture medium and clinical biofluids such as serum, saliva, ascitic fluid, and cerebrospinal fluid. We believe that ExoNANO might pave the way for the rapid isolation and quantitative analysis of exosomes for routine clinical applications.