Current paper-based potentiometric ion-sensing platforms are planar devices used for clinically relevant ions. These devices,h owever,h ave not been designed for the potentiometric biosensing of proteins or small molecule analytes.At hree-dimensional origami paper-based device,i n which as olid-contact ion-selective electrode is integrated with an all-solid-state reference electrode,i sd escribed for the first time.T he device is made by impregnation of paper with appropriate bioreceptors and reporting reagents on different zones.Byfolding and unfolding the paper structures,versatile potentiometric bioassays can be performed. AUSB-controlled miniaturized electrochemical detector can be used for simple and in situ measurements.U sing butyrylcholinesterase as am odel enzyme,t he device has been successfully applied to the detection of enzyme activities and organophosphate pesticides involved in the enzymatic system as inhibitors.T he proposed 3D origami paper device allows the potentiometric biosensing of proteins and small molecules in as imple, portable,and cost-effective way.