In this study, a paper-based sensor, combined with a visual distance-readout method, was developed to determine glucose in fruit samples based on the glucose oxidase-mediated sodium alginate gelation. The type of filter paper, the concentration of sodium alginate and the enzymatic reaction conditions were systematically investigated. Under optimal conditions, the increase in diffusion diameter showed a good linear relationship with glucose concentration between 1.4–7.0 mM, and the limit of quantification was 1.4 mM. Finally, the applicability of the proposed strategy was successfully verified by measuring glucose concentrations in fruit samples. The results generated by the developed paper-based sensor were in good agreement with the results obtained from a glucose assay kit. The recoveries were 91.8%–99.1%. In short, the present study developed a simple, low-cost and efficient method for assessing fruit quality and for guiding fruit intake for diabetic patients, especially in remote or resource-limited regions.