Wearable biosensors that can monitor health conditions are currently attracting attention. In this study, we investigated a method to fabricate a glucose biosensor by heat transfer printing. A screen-printed glucose oxidase-modified electrode formed on a polyethylene terephthalate (PET) substrate was transferred onto a nonwoven fabric. Conditions for thermal transfer while retaining enzyme activity were investigated, and electrochemical evaluation of the glucose biosensor was conducted. Enzyme-catalyzed reactions were found to readily occur on the electrode surface after heat transfer printing. A linear relationship was obtained in the glucose concentration range from 0.1 to 20 mmol dm−3. In general, the glucose concentration in urine is 3–6 mmol dm−3 in healthy persons and 7 mmol dm−3 or higher in diabetics. This fabrication method is expected to be widely applicable to future biosensors for monitoring urine sugar in diapers and other products.