A facile preparation method of a disposable capillary biosensor immobilizing a plasticized poly(vinyl chloride) (PVC) membrane ion-selective optode containing a lipophilic pH indicator dye and an ionophore, and a dissolvable poly(ethylene glycol) (PEG) membrane containing an enzyme is developed. The method involves the sequential introduction of membrane cocktail solution followed by air introduction. Repeating this process allowed facile preparation of a capillary biosensor comprised of two layer structure, which can be used for single-step biosensing just by sipping the sample with capillary action. Here a urea biosensor was prepared as a proof-of-concept.