Exosomes as nanosized biomarkers hold great potential for the diagnosis of cancer. However, the low concentration of cancer-derived exosomes present in biofluids makes early diagnosis strenuous. Here, we developed a fluorescent biosensing platform, namely a dual signal amplification, for the ultrasensitive detection of leukemia cell-derived exosomes. The protocol consists of three steps: first, leukemia-derived exosomes containing CD63 and nucleolin were captured by anti-CD63 antibody modified magnetic bead conjugates (MB-CD63); then, a DNA primer comprising a nucleolin-recognition aptamer (AS1411) was applied to bind the exosomes which further initiated a rolling circle amplification (RCA) reaction to generate many repeat sequences for hybridization with gold nanoparticle (GNP)-DNA-fluorescent dye (FAM) conjugates (GNP-DNA-FAM); finally, nicking endonuclease (Nb·BbvCI) assisted target recycling was introduced. As a result, FAM was released from GNP-DNA-FAM conjugates, transformed from the quenching state to the emission state and thus fluorescence signals continuously accumulated. With this dual signal amplification platform, as low as 1 × 102 particles per μL exosomes could be detected. Furthermore, we have successfully applied this method for the detection of exosomes in spiked serum samples, indicating a promising tool for clinical application.