Silk cocoons were used as a bioelectrode for the wearable electrochemical sensors of sweat lactate via carbonization and the in situ electrodeposition of platinum nanoparticles (PtNPs) and reduced graphene oxide (rGO), followed by lactate oxidase immobilization. Scanning electron microscopy, atomic force microscopy, and x-ray photoelectron microscopy confirmed that PtNPs/rGO were deposited on the carbonized silk cocoon surfaces, characterized via the physical and chemical alterations of silk cocoons. The electrocatalytic activity of PtNPs and the high surface area and functionality of rGO enhanced the electrochemical sensitivity of the sensor in lactate detection. This biosensor detected sweat lactate selectively in a range of 0–25 mM with a limit of detection of 0.07 mM, which is sufficient to distinguish between normal individuals and muscle fatigue-prone patients at a cut-off sweat lactate level of 12.5 mM. This biosensor was applied for sweat lactate detection and validated through laser desorption–ionization mass spectrometry with satisfactory results. This bioelectrode exhibits cytocompatibility with non-irritation and non-allergy to human skin, highlighting its application as a wearable lactate biosensor for self-monitoring of muscle fatigue.