A new signal amplification strategy based on DNA hybridization–dehybridization reaction on the surface of magnetic submicrobeads (MSBs) for fluorescence detection of ultrasensitive DNA was developed. In this strategy, MSBs modified with probe DNA (DNAp-MSBs) were bound to target DNA (t-DNA) (with a ratio of 1 : 1) captured to a substrate. The DNAp-MSBs were released from the substrate via DNA dehybridization and then hybridized with Cy5-labeled detection DNA (Cy5-DNAd). After the Cy5-DNAd and DNAp-MSBs were separated by dehybridization, the Cy5-DNAd was collected. The DNAp-MSBs were then hybridized with other Cy5-DNAd to initiate the next hybridization–dehybridization round. This recycling of the hybridization–dehybridization process on the surface of the DNAp-MSBs was repeated multiple times to accumulate Cy5-DNAd. Finally, fluorescence intensity of the collected Cy5-DNAd was measured. Using this strategy, the limit of detection for determination of t-DNA was 8.5 × 10⁻¹⁵ mol L⁻¹ for 11 cycles. The ultrasensitive assay was used to quantify ribosomal protein, large, P2 (RPLP2) mRNA in human breast cancer cells.