Colorimetric　methods　to　detect　glucose　are　promising　for　the　wide　applications　in　the　assays　of　diabetes　diagnosis,　environments　and　foods,　but　traditional　colorimetric　methods　for　glucose　detection　are　usually　limited　in　the　use　of　peroxidase　or　the　complex　chemical　synthesis　of　nanomaterials,　and　improving　sensitivity　remains　a　challenging　task.　In　this　paper,　based　on　the　conformational　switch　of　i-motif　DNA　triggered　by　the　variation　of　pH　value　resulting　from　the　oxidation　of　glucose,　a　facile　colorimetric　and　ratiometric　biosensor　for　selective　and　sensitive　detection　of　glucose　is　propounded.　In　such　an　assay　protocol,　glucose　was　detected　expediently　by　the　color　difference　between　free　methylene　blue　(MB)　and　MB　bound　with　a　cytosine-rich　single-stranded　DNA　without　using　any　peroxidase　or　nanomaterials.　Delightedly,　the　assay　demonstrated　a　wide　linear　response　for　the　glucose　concentration　in　a　range　from　5　to　800　µmol/L　with　a　low　detection　limit　of　1.43　µmol/L　(3　s　/　slope).　Besides,　this　biosensor　was　applied　in　detecting　glucose　in　human　serum　samples　successfully,　suggesting　its　potential　of　applying　in　the　healthcare　of　monitoring　glucose　with　a　cheap,　convenient　and　enzyme-free　method.　©　2020