Circulating　cell-free　DNA　(cfDNA)　is　known　to　be　an　attractive　biomarker　for　liquid　biopsy.　In　this　work,　a　rapid　and　sensitive　biosensor　was　achieved　for　cfDNA　detection　based　on　CRISPR-associated　nuclease　12a　(CRISPRCas12a)　and　single　nanoparticle　dark-field　microscopy　(DFM)　imaging.　The　biosensor　employed　gold　nanoparticles　(AuNPs)　as　signal　source,　DFM　as　readout　system,　and　Meanshift　algorithms　as　the　image　processing　systems,　respectively.　The　presence　or　absence　of　cfDNA　caused　the　different　existing　states　(monomer　or　aggregated　state)　of　AuNPs.　AuNPs　monomers　could　be　effectively　distinguished　from　the　aggregated　ones　under　DFM　since　the　AuNPs　aggregation　could　induced　the　green-to-yellow　or　green-to-red　changing　of　scattering　color.　The　monomer　ratio　easily　obtained　by　Meanshift　and　partial　least-square　(PLS)　algorithms　was　used　for　quantitative　analysis.　Using　this　strategy,　breast　cancer　gene-1　(BRCA-1),　a　representative　of　cfDNA　biomarker　for　breast　cancer　could　be　measured　with　high　sensitivity　in　40　min　and　has　a　low　detection　limit　of　0.081　fM.　It　is　hoped　that　this　nucleic　acid　amplification-free　sensor　could　be　also　utilized　to　detect　other　nucleic　acid　biomarkers,　and　thus,　provides　a　novel　strategy　for　biomedical　image　analysis.