There is a growing demand for inexpensive and user-friendly detection technologies that can identify various biomarkers in a sustainable manner. Microfluidic thread-based analytical devices (μTADs) are rapidly growing because of their biocompatibility, flexibility, and absence of complex treatments. Additionally, μTADs are highly compact, allowing for the use of small sample volumes, with the potential to compete with low-cost paper in point-of-care (POC) applications. Here, we present a chemiluminescent thread-based device for user-friendly detection of lactate in sweat, which is an indicator of performance in athletic training programs, and it can provide vital information connected to ischemia and insufficient oxidative metabolism. Lactate oxidase (LOx)-catalyzed reaction coupled with the luminol/H₂O₂/horseradish peroxidase (HRP) system was exploited by immobilizing substrate and enzymes on different sides of the same thread, avoiding undesired premature reactions. Small-volume samples (2 µL) were used on an optimized “X-shaped” device with an incubation time of 5 min. Different supports were tested from the perspective of wearability of the biosensing thread, including polypropylene, mother of pearl buttons and, to improve sustainability, grape and apple skin obtained from agro-food waste. Smartphone detection allowed a simple and quantitative readout for the end-user. Under optimized conditions, the biosensor achieved detection limits of 0.27, 0.24, and 0.15 mM for lactate spiked in artificial sweat samples at pH 5.5, 6.5, and 8.0, respectively, showing promising applications for POC applications.