Fatigue causes deleterious effects to physical and mental health of human being and may cause loss of lives. Therefore, the adverse effects of fatigue on individuals and the society are massive. With the ever‐increasing frequency of overtraining among modern military and sports personnel, timely, portable and accurate fatigue diagnosis is essential to avoid fatigue‐induced accidents. However, traditional detection methods require complex sample preparation and blood sampling processes, which cannot meet the timeliness and portability of fatigue diagnosis. With the development of flexible materials and biosensing technology, wearable biosensors have attracted increased attention to the researchers. Wearable biosensors collect biomarkers from noninvasive biofluids, such as sweat, saliva, and tears, followed by biosensing with the help of biosensing modules continuously and quantitatively. The detection signal can then be transmitted through wireless communication modules that constitute a method for real‐time understanding of abnormality. Recent developments of wearable biosensors are focused on miniaturized wearable electrochemistry and optical biosensors for metabolites detection, of which, few have exhibited satisfactory results in medical diagnosis. However, detection performance limits the wide‐range applicability of wearable fatigue diagnosis. In this article, the application of wearable biosensors in fatigue diagnosis has been discussed. In fact, exploration of the composition of different biofluids and their potential toward fatigue diagnosis have been discussed here for the very first time. Moreover, discussions regarding the current bottlenecks in wearable fatigue biosensors and the latest advancements in biochemical reaction and data communication modules have been incorporated herein. Finally, the main challenges and opportunities were discussed for wearable fatigue diagnosis in the future.