A　novel　electrochemical　immunosensor　was　designed　for　sensitive　detection　of　mycotoxins　(aflatoxin　B1,　AFB1,　used　in　this　case)　by　coupling　proximity　ligation　assay-induced　conformation　switch　of　hairpin　DNA　with　the　antigen-antibody　reaction.　The　assay　was　carried　out　by　anti-AFB1　antibody-conjugated　DNA1　(mAb-DNA1),　AFB1-BSA-labeled　DNA2　(AFB1-DNA2)　and　hairpin　DNA.　Each　hairpin　included　a　stem　of　6　base　pairs　and　a　6　nucleotide　(nt)　loop　with　the　labelled　ferrocene　tag　at　the　3′　end,　which　was　immobilized　on　the　electrode　via　self-assembly　of　the　terminal　thiol　moiety　at　the　5′　end.　The　proximity　ligation　assay　was　carried　out　via　the　specific　antigen-antibody　reaction　between　mAb-DNA1　and　AFB1-DNA2　to　form　an　omega-like　DNA　junction.　Thereafter,　the　junction　hybridized　with　hairpin　DNA　to　open　the　probe,　thus　resulting　in　the　ferrocene　tag　far　away　from　the　electrode　for　the　decreasing　of　the　redox　current.　Upon　target　AFB1　introduction,　the　competitive-type　immunoassay　was　executed　between　the　analyte　and　AFB1-DNA2　for　mAb-DNA1.　Under　the　optimal　conditions,　the　electrochemical　signal　was　indirectly　proportional　to　target　AFB1　concentration,　and　allowed　the　detection　of　target　AFB1　at　a　concentration　as　low　as　3.2　pg　mL−1.　Our　strategy　also　exhibited　high　selectivity,　good　reproducibility　and　precision.　Importantly,　the　accuracy　of　this　methodology　was　validated　for　analysis　of　spiked　or　naturally　contaminated　peanut　samples,　giving　results　matched　well　with　those　obtained　from　commercialized　available　AFB1　ELISA　kit.　©　2016　WILEY-VCH　Verlag　GmbH　&　Co.　KGaA,　Weinheim