The monosaccharide 1,5-anhydroglucitol (1,5-AG) is a known indicator of glucose levels. Conventional 1,5-AG quantification methods with enzyme-based sensors using pyranose oxidase (PROD) require elimination of interference from the sample (a laborious and time-consuming process), as PROD cannot distinguish 1,5-AG from other sugars. We developed a one-step paper-based sensor for detecting 1,5-AG using glucose oxidase, catalase, and mutarotase that eliminates excess glucose, which interferes with 1,5-AG detection. This sensor consists of two compartments for the quantification of glucose and 1,5-AG and reflects the concentration of these targets after reaction with water or spiked human urine. The limit of detection of the sensor was 0.9 mg dL-1 for glucose and 3.2 μg mL-1 for 1,5-AG.