Herein,　a　novel　signal-on　photoelectrochemical　(PEC)　biosensor　with　nearly　zero　background　noise　(ZBN)　was　first　fabricated　to　determine　the　presence　of　organophosphorus　pesticide　based　on　in　situ　formation　of　DNA-templated　Ag2S　photoactive　materials,　accompanied　by　hybridization　chain　reaction　(HCR)　signal　amplification.　The　capture　probe　(S1)　on　the　gold　nanoparticle-modified　electrode　can　hybridize　with　the　aptamer　molecule　to　generate　a　simple　PEC　biosensor.　In　the　presence　of　a　target　molecule,　the　aptamer　molecule　is　released　on　the　double-stranded　DNA　(dsDNA)-modified　PEC　biosensor.　Meanwhile,　the　capture　probe　remains　on　the　electrode　and　can　open　the　DNA　hairpins　(H1,　H2)　which　are　rich　in　cytosine,　to　trigger　the　HCR　reaction.　The　rich　＇C＇　strands　are　uncovered　after　formation　of　a　long　dsDNA　polymer　strand,　which　can　assemble　multiple　silver　ions　(Ag+)　by　means　of　by　C–Ag+–C　chelation.　Then,　a　large　number　of　Ag2S　can　be　generated　by　challenging　with　S2−　solution,　producing　a　satisfactory　photocurrent　signal.　The　photoactive　material　is　formed　in　situ,　which　eliminates　the　laborious　operation.　Moreover,　the　signal　can　be　highly　amplified　with　nearly　zero　background　noise　and　HCR　signal　amplification.　Under　optimal　conditions,　the　ZBN　aptasensor　exhibited　high　sensitivity　and　selectivity,　with　a　low　detection　limit　of　2 pg mL−1　for　malathion.　Importantly,　the　sensing　platform　can　also　be　applied　to　determine　the　presence　of　malathion　in　real　samples.　Graphical　abstract:　[Figure　not　available:　see　fulltext.]　©　2021,　Springer-Verlag　GmbH　Germany,　part　of　Springer　Nature.