The　pH　meter,　one　of　the　most　common　sensors　for　laboratory　measurements,　has　the　advantages　of　excellent　sensitivity,　low　price,　high　reliability,　and　ease　of　use.　In　this　study,　the　high　sensitivity　of　the　pH　meter　was　coupled　with　the　high　selectivity　of　immunosorbent　assay　to　develop　a　simple　and　portable　biosensor　for　aflatoxin　B1　(AFB1)　determination.　Urease　was　first　labelled　on　the　AFB1　antigen,　in　the　presence　of　AFB1;　the　target　competed　with　the　urease-labeled　AFB1　antigen　to　be　captured　by　the　antibody.　Urease　could　catalyse　the　hydrolysis　of　urea　into　ammonia　and　induce　the　pH　increase　of　the　solution;　the　change　could　easily　be　detected　using　a　handheld　pH　meter　or　pH-sensitive　dye.　Under　optimized　conditions,　the　pH　value　enhancement　had　a　linear　relationship　with　the　logarithm　of　the　concentration　of　AFB1　in　the　range　of　0.62　to　23.42　ng　mL-1.　The　proposed　biosensor　has　been　applied　to　detect　AFB1　in　real　samples　with　satisfactory　results.　©　2018　The　Royal　Society　of　Chemistry.