A　new　homogeneous　electrochemical　immunosensing　platform　was　designed　for　sensitive　detection　of　aflatoxin B1　(AFB1)　in　foodstuff.　The　system　consisted　of　anti-AFB1　antibody　labeled　DNA1　(Ab-DNA1),　AFB1–bovine　serum　albumin　(BSA)-conjugated　DNA2　(AFB1-DNA2),　and　methylene　blue　functionalized　hairpin　DNA.　Owing　to　a　specific　antigen–antibody　reaction　between　anti-AFB1　and　AFB1–BSA,　the　immunocomplex　formed　assisted　the　proximity　hybridization　of　DNA1　with　DNA2,　thus　resulting　in　the　formation　of　an　omega-like　DNA　junction.　Thereafter,　the　junction　opened　the　hairpin　DNA　to　construct　a　new　double-stranded　DNA,　which　could　be　readily　cleaved　by　exonuclease III　to　release　the　omega-like　DNA　junction　and　methylene　blue.　The　dissociated　DNA　junction　could　repeatedly　hybridize　with　residual　hairpin　DNA　molecules　with　exonuclease III-based　isothermal　cycling　amplification,　thereby　releasing　numerous　free　methylene　blue　molecules　into　the　detection　solution.　The　as-produced　free　methylene　blue　molecules　could　be　captured　by　a　negatively　charged　indium　tin　oxide　electrode,　each　of　which　could　produce　an　electronic　signal　within　the　applied　potentials.　On　introduction　of　target　AFB1,　the　analyte　competed　with　AFB1-DNA2　for　the　conjugated　anti-AFB1　on　the　Ab-DNA1,　subsequently　decreasing　the　amount　of　omega-like　DNA　junctions　formed,　hence　causing　methylene　blue　labeled　hairpin　DNA　to　move　far　away　from　the　electrode　surface.　Under　optimal　conditions　the　detectable　electrochemical　signal　decreased　with　increasing　amount　of　target　AFB1　in　a　dynamic　working　range　of　0.01–30 ng mL-1　with　a　detection　limit　of　4.8 pg mL-1.　In　addition,　the　precision　and　reproducibility　of　this　system　were　acceptable.　Finally,　the　method　was　further　evaluated　for　analysis　of　naturally　contaminated　or　AFB1-spiked　peanut　samples,　giving　results　that　matched　well　with　those　obtained　with　a　commercial　AFB1　ELISA　kit.　©　2016,　Springer-Verlag　Berlin　Heidelberg.