A　sensitive　homogeneous　electrochemiluminescence　(ECL)　biosensor　for　flap　endonuclease　1　(FEN1)　detection　was　developed　by　combining　highly　sensitive　ECL　detection,　high　efficiency　of　branched　hybridization　chain　reaction　(BHCR)　amplification,　a　convenient　homogeneous　strategy,　and　simple　ultrafiltration　separation.　Magnetic　beads　were　first　modified　with　well-designed　double　flap　DNAs　containing　5＇-flaps.　In　the　presence　of　FEN1,　the　5＇-flap　can　be　cleaved,　and　a　large　amount　of　single-stranded　DNA　can　be　produced,　which　can　be　separated　easily　from　the　double-flap　DNA-modified　beads　by　a　magnet.　Then,　the　cleaved　5＇-flap　can　be　used　to　initiate　BHCR　amplification　to　produce　a　large　amount　of　long-strand　dsDNA.　Ru(phen)(3)(2+)　can　insert　dsDNA　to　form　Ru-dsDNAs,　which　can　be　easily　separated　from　the　main　solution　through　ultrafiltration.　The　ECL　signal　from　the　separated　Ru-dsDNAs　has　a　good　linear　relationship　with　the　logarithm　of　the　FEN1　concentration　ranging　from　6.5　x　10(-2)　similar　to　6.5　x　10(3)　U/L　with　a　detection　limit　of　2.2　x　10(-2)　U/L.　The　proposed　biosensor　was　used　to　evaluate　FEN1　activity　in　real　samples　with　satisfactory　results.