Chloroethylnitrosoureas　(CENUs)　were　an　important　family　of　anticancer　agents　in　the　clinical　treatment　of　human　malignancies.　Several　lines　of　evidences　indicated　that　their　cytotoxicity　was　related　to　the　interstrand　crosslink　of　DNA.　In　this　study,　a　method　for　the　determination　of　DNA　crosslink　was　established　using　real-time　fluorescence　quantitative　PCR.　The　DNA　crosslink　ratio　induced　by　two　CENUs,　Semustine　(Me-CCNU)　and　Carmustine　(BCNU)　were　quantitatively　analyzed　and　compared.　The　results　showed　that　BCNU　and　Me-CCNU　exhibit　different　crosslink　trend　of　double　strand　DNA.　Evagreen,　a　fluorescent　dye,　was　used　as　the　probe　in　the　assay.　This　study　provided　direct　evidence　that　real-time　fluorescence　quantitative　PCR　(qPCR)　was　a　more　convenient,　efficient　and　stable　method　to　detect　DNA　crosslink.