Most　DNA-based　electrochemiluminescence　(ECL)　biosensors　are　established　through　the　self-assembly　of　thiolated　single-stranded　DNA　(ssDNA)　probes　on　the　Au　electrode　surface.　Because　of　this　random　assembly　process,　a　significant　discrepancy　exists　in　the　distribution　of　a　modified　DNA　film　on　different　electrodes,　which　greatly　affects　the　reproducibility　of　a　biosensor.　In　this　study,　a　porous　bovine　serum　albumin　(BSA)　layer　was　first　modified　on　the　electrode　surface,　which　can　improve　the　position　distribution　and　spatial　orientation　of　the　self-assembly　ssDNA　probe.　It　was　then　coupled　with　hyperbranched　rolling　circle　amplification　to　develop　the　high-reproducibility-and-sensitivity　ECL　biosensor　for　human　papillomavirus　16　E6　and　E7　oncogene　detection.　In　the　presence　of　the　target　DNA,　the　surface　of　the　electrode　accumulates　abundant　amplified　products　through　reaction,　which　contain　double-stranded　DNA　(dsDNA)　fragments　of　different　lengths,　followed　by　plentiful　dichlorotris　(1,10-phenanthroline)　ruthenium(II)　hydrate　(Ru(phen)32+,　acting　as　an　ECL　indicator)　insertion　into　grooves　of　dsDNA　fragments,　and　a　strong　signal　can　be　detected.　There　is　a　linear　relationship　between　the　signal　and　the　target　concentration　range　from　10　fM　to　15　pM,　and　the　detection　limit　is　7.6　fM　(S/N　=　3).　After　the　BSA　modification　step,　the　relative　standard　deviation　was　reduced　from　9.20　to　3.96%,　thereby　achieving　good　reproducibility.　The　proposed　ECL　strategy　provides　a　new　method　for　constructing　high-reproducibility-and-sensitivity　ECL　biosensors.　©