A highly sensitive and specific colorimetry-based rolling circle amplification (RCA) assay has been successfully developed as a method for the effective detection of H1N1 DNA. Specific oligonucleotide and reporter primer probes were designed together with a circular template, and the oligonucleotide probes were attached to the surfaces of magnetic beads (MBs) to form functional MB–DNA conjugates as capture probes for the target H1N1 DNA molecules. Together with the addition of DNA targets and reporter primer probes to the MB–DNA conjugates, sandwiched hybrids were formed. The initiation of RCA amplification using the circular template in the presence of phi29 polymerase allowed for the amplification of a large number of repeat sequences of the single-stranded (ss)-DNA product. This RCA product accumulated gold nanoparticles (AuNPs), resulting in a colorimetric change that could be viewed by the naked eye or detected using UV-vis spectroscopy. According to this method, H1N1 DNA could be detected at the 1 pmol L⁻¹ level. This platform exhibited design convenience, simplicity, and cost-effectiveness, and could be used to provide a new diagnostic assay for H1N1, and other infectious diseases.