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Wearable sweat biosensors have shown great progress in noninvasive, in situ, and continuous health monitoring to demonstrate individuals’ physiological states. Advances in novel nanomaterials and fabrication methods promise to usher in a new era of wearable biosensors. Here, we introduce a threedimensional (3D)-printed flexible wearable health monitor fabricated through a unique one-step continuous manufacturing process with self-supporting microfluidic channels and novel single-atom catalyst-based bioassays for measuring the sweat rate and concentration of three biomarkers. Direct ink writing is adapted to print the microfluidic device with self-supporting structures to harvest human sweat, which eliminates the need for removing sacrificial supporting materials and addresses the contamination and sweat evaporation issues associated with traditional sampling methods. Additionally, the pick-and-place strategy is employed during the printing process to accurately integrate the bioassays, improving manufacturing efficiency. A single-atom catalyst is developed and utilized in colorimetric bioassays to improve sensitivity and accuracy. A feasibility study on human skin successfully demonstrates the functionality and reliability of our health monitor, generating reliable and quantitative in situ results of sweat rate, glucose, lactate, and uric acid concentrations during physical exercise.