Target-induced　differences　in　the　electrostatic　interactions　between　methylene　blue　(MB)　and　indium　tin　oxide　(ITO)　electrode　surface　was　firstly　employed　to　develop　a　homogeneous　electrochemical　biosensor　for　flap　endonuclease　1　(FEN1)　detection.　In　the　absence　of　FEN1,　the　positively　charged　methylene　blue　(MB)　is　free　in　the　solution　and　can　diffuse　onto　the　negatively　charged　ITO　electrode　surface　easily,　resulting　in　an　obvious　electrochemical　signal.　Conversely,　with　the　presence　of　FEN1,　a　5＇-flap　is　cleaved　from　the　well-designed　flapped　dumbbell　DNA　probe　(FDP).　The　remained　DNA　fragment　forms　a　closed　dumbbell　DNA　probe　to　trigger　hyperbranched　rolling　circle　amplification　(HRCA)　reaction,　generating　plentiful　dsDNA　sequences.　A　large　amount　of　MB　could　be　inserted　into　the　produced　dsDNA　sequences　to　form　MB-dsDNA　complexes,　which　contain　a　large　number　of　negative　charges.　Due　to　the　strong　electrostatic　repulsion　between　MB-dsDNA　complexes　and　the　ITO　electrode　surface,　a　significant　signal　drop　occurs.　The　signal　change　(ΔCurrent)　shows　a　linear　relationship　with　the　logarithm　of　FEN1　concentration　from　0.04　to　80.0　U/L　with　a　low　detection　limit　of　0.003　U/L　(S/N　=　3).　This　study　provides　a　label-free　and　homogeneous　electrochemical　platform　for　evaluating　FEN1　activity.