Exosomes as fluid biomarkers hold great promise for noninvasive cancer diagnosis. However, a method for the rapid and convenient detection of exosomes is still a challenge because current analysis processes involve multiple steps and yield low sensitivity. Here, we developed a wash-free fluorescent biosensor for the rapid and sensitive quantification of exosomes by combining aptamer and nicking endonuclease (Nb·BbvCI). In this system, an aptamer–trigger complex was used as the recognition element; the trigger probe could be released, and it hybridized with gold nanoparticles (GNPs)–DNA–FAM conjugates, thereby resulting in Nb·BbvCI-assisted target recycling. As a result, our method allowed the quantification of exosomes with lower analysis time by using a cocktail containing an aptamer–trigger complex, Nb·BbvCI, and GNPs–DNA–FAM. A high sensitivity with a limit of detection (LOD) of 1.0 × 10⁴ particles per μL could be achieved. Besides, this biosensor exhibited potential application for the quantification of exosomes in human plasma, facilitating the development of exosome-based noninvasive cancer diagnosis.