Argonaute 2 (Ago2) is an essential component of the RNA-induced silencing complex (RISC) and it participates in diverse physiological processes, while dysregulation of Ago2 activity is closely linked to a variety of human diseases including cancers. The reported Ago2 assays often suffer from laborious procedures, complicated reaction schemes, and unsatisfactory sensitivity. Herein, we develop a new gold nanoparticle (AuNP)-based single-molecule biosensor for simple and sensitive detection of Ago2 activity. The Ago2-responsive AuNP nanoprobe is constructed through the self-assembly of multiple Cy5-labeled signal probes onto the AuNP, in which the Cy5 fluorescence is efficiently quenched by the AuNP. Target Ago2 can bind with guide RNA to form an active RISC, inducing the cyclic cleavage of the signal probes and the release of Cy5 moieties from the AuNP nanoprobe. The released Cy5 molecules can be simply quantified by single-molecule counting. This single-molecule biosensor enables detection of Ago2 activity with the involvement of only a single AuNP nanoprobe, eliminating the use of any extra antibodies and protein enzymes. This single-molecule biosensor achieves good specificity and high sensitivity with a detection limit of 9.1 pM, and it can be exploited for the screening of Ago2 inhibitors, Ago2 kinetic analysis, and the imaging of intracellular Ago2 activity in live cells, holding great promise in Ago2-related biomedical research and clinical diagnosis.