The　lack　of　large-scale　human　papillomavirus　(HPV)　DNA　screening　is　a　major　contributor　to　the　high　incidence　and　mortality　of　cervical　cancer　in　economically　undeveloped　areas.　The　development　of　sensitive,　rapid,　and　low-cost　screening　techniques　is　urgently　needed.　Here,　DNA　nanoflowers　encapsulating　glucose　oxidase　and　horseradish　peroxidase　(GHDFs)　were　synthesized　by　one-pot　rolling　circle　amplification,　and　then　the　GHDFs　were　used　as　the　cargo　of　DNA　hydrogel　and　applied　for　HPV　DNA　detection.　When　target　DNA　was　present,　the　DNA　hydrogel　cross-linking　structure　was　disrupted,　releasing　GHDFs,　which　then　catalysed　the　oxidation　of　glucose　and　tetramethylbenzidine　in　a　cascade,　generating　a　significant　electrical　signal.　Signal　intensity　had　a　linear　relationship　with　the　logarithm　of　target　DNA　concentration　in　the　range　of　10　fM-1　nM　with　a　detection　limit　of　3.76　fM.　The　detection　time　of　the　proposed　biosensor　was　25　min,　which　was　suitable　for　large-scale　HPV　DNA　screening　in　economically　underdeveloped　areas　and　provides　a　blueprint　for　the　detection　of　other　DNA　of　interest.　©　2022　Elsevier　B.V.