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In this work, a label-free colorimetric assay was developed for the determination of urine glucose using smartphone ambient-light sensor (ALS). Using horseradish peroxidase—hydrogen peroxide—3,3′,5,5′-tetramethylbenzidine (HRP-H₂O₂-TMB) colored system, quantitative H₂O₂ was added to samples to-be-determined for deepest color. The presence of glucose oxidase in urine led to the formation of H₂O₂ and the reduction of TMB_red. As a result of this, the color of the urine faded and the solution changed from deep blue to light blue. We measured the illuminance of the transmitted light by a smartphone ambient light sensor, and thereby color changes were used to calculate the content of urine glucose. After method validation, this colorimetric assay was practically applied for the determination of urine samples from diabetic patients. Good linearity was obtained in the range of 0.039−10.000 mg/mL (R² = 0.998), and a limit of detection was 0.005 mg/mL. Our method was had high accuracy, sensitivity, simplicity, rapidity, and visualization, providing a new sensor to be potentially applicable for point-of-care detection of urine glucose.