This study reports a microfluidic chip-based wearable colorimetric sensor for detecting sweat glucose. The device consisted of five microfluidic channels branching out from the center and connected to the detection microchambers. The microchannels could route the sweat excreted from the epidermis to the microchambers, and each of them was integrated with a check valve to avoid the risk of the backflow of the chemical reagents from the microchamber. The microchambers contained the pre-embedded glucose oxidase (GOD)–peroxidase–o-dianisidine reagents for sensing the glucose in sweat. It was found that the color change caused by the enzymatic oxidation of o-dianisidine could show a more sensitive response to the glucose than that of the conventional GOD–peroxidase–KI system. This sensor could perform five parallel detections at one time. The obtained linear range for sweat glucose was 0.1–0.5 mM with a limit of detection of 0.03 mM. The sensor was also used to detect the glucose in sweat samples from a group of subjects engaged in both fasting and postprandial trials. The results showed that our wearable colorimetric sensor can reveal the subtle differences existing in the sweat glucose concentration after the fasting and the oral glucose uptake.