A　new　photoluminescence　(PL)　enzyme　immunoassay　was　designed　for　sensitive　detection　of　aflatoxin　B1　(AFB1)　via　an　innovative　enzyme　substrate,　6-aza-2-thiothymine-stabilized　gold　nanocluster　(AAT-AuNC)　with　L-arginine.　The　enzyme　substrate　with　strong　PL　intensity　was　formed　through　supramolecular　host-guest　assembly　between　guanidine　group　of　L-arginine　and　AAT　capped　on　the　surface　of　AuNC.　Upon　arginase　introduction,　the　captured　L-arginine　was　hydrolyzed　into　ornithine　and　urea,　thus　resulting　in　the　decreasing　PL　intensity.　Based　on　this　principle,　a　novel　competitive-type　immunoreaction　was　first　carried　out　on　AFB1-bovine　serum　albumin　(AFB1-BSA)　conjugate-coated　microplate,　using　arginase-labeled　anti-AFB1　antibody　as　the　competitor.　Under　the　optimum　conditions,　the　PL　intensity　increased　with　the　increment　of　target　AFB1,　and　allowed　the　detection　of　the　analyte　at　concentrations　as　low　as　3.2　pg　mL−1　(ppt).　Moreover,　L-arginine-AAT-AuNC-based　PL　enzyme　immunoassay　afforded　good　reproducibility　and　acceptable　specificity.　In　addition,　the　accuracy　of　this　methodology,　referring　to　commercial　AFB1　ELISA　kit,　was　evaluated　to　analyze　naturally　contaminated　or　spiked　peanut　samples,　giving　well-matched　results　between　two　methods,　thus　representing　a　useful　scheme　for　practical　application　in　quantitative　monitoring　of　mycotoxins　in　foodstuff.　©　2018　Elsevier　B.V.