A　new　strategy　for　development　of　electrochemical　DNA　biosensor　based　on　site-specific　DNA　cleavage　of　restriction　endonuclease　and　using　quantum　dots　as　reporter　was　reported　in　this　paper.　The　biosensor　was　fabricated　by　immobilizing　a　capture　hairpin　probe,　thiolated　single　strand　DNA　labeled　with　biotin　group,　on　a　gold　electrode.　BfuCI　nuclease,　which　is　able　to　specifically　cleave　only　double　strand　DNA　but　not　single　strand　DNA,　was　used　to　reduce　background　current　and　improve　the　sensitivity.　We　demonstrated　that　the　capture　hairpin　probe　can　be　cleaved　by　BfuCI　nuclease　in　the　absence　of　target　DNA,　but　cannot　be　cleaved　in　the　presence　of　target　DNA.　The　difference　before　and　after　enzymatic　cleavage　was　then　monitored　by　electrochemical　method　after　the　quantum　dots　were　dissolved　from　the　hybrids.　Our　results　suggested　that　the　usage　of　BfuCI　nuclease　obviously　improved　the　sensitivity　and　selectivity　of　the　biosensor.　We　successfully　applied　this　method　to　the　sequence-selective　discrimination　between　perfectly　matched　and　mismatched　target　DNA　including　a　single-base　mismatched　target　DNA,　and　detected　as　low　as　3.3×10-14M　of　complementary　target　DNA.　Furthermore,　our　above　strategy　was　also　verified　with　fluorescent　method　by　designing　a　fluorescent　molecular　beacon　(MB),　which　combined　the　capture　hairpin　probe　and　a　pair　of　fluorophore　(TAMRA)　and　quencher　(DABCYL).　The　fluorescent　results　are　consistent　with　that　of　electroanalysis,　further　indicating　that　the　proposed　new　strategy　indeed　works　as　we　expected.　©　2010　Elsevier　B.V.