Alcohol misuse has become a major public health concern worldwide. The development of an accurate, non-invasive, and cost-effective method for real-time alcohol detection would have important applications for monitoring and preventing alcohol misuse. This paper proposes a novel wearable biosensor for real-time alcohol detection in sweat. The biosensor uses alcohol oxidase immobilized on a screen-printed electrode to amperometrically detect ethanol in sweat samples. An artificial sweat solution was used to optimize the sensor design and evaluate its analytical performance. The optimized biosensor demonstrated a linear response to ethanol in the concentration range of 0.1–1.0 g/dL, with a limit of detection of 0.02 g/dL. On-body trials of the wearable biosensor provided rapid measurements that strongly correlated with blood alcohol content. This approach could offer a convenient solution for continuous, non-invasive alcohol monitoring to help reduce alcohol misuse and promote healthy behaviors. Further research on long-term use, alternate electrode geometries, and fully integrated wearable designs is warranted.