Oxidative stress is widely recognized as a pivotal factor contributing to numerous Central Nervous System (CNS) ailments. The concentrations of hydrogen peroxide (H2O2) and phosphorylated proteins within the human body serve as crucial indicators of oxidative stress. As such, the real-time monitoring of H2O2 and phosphorylated proteins in sweat is vital for the early identification, diagnosis, and management of diseases linked to oxidative stress. In this context, we present a novel microfluidic wearable electrochemical sensor by modifying the electrode with Prussian blue (PB) and loading sulfur-rich vacancy-containing molybdenum disulfide (MoS2-X) onto Multi-walled carbon nanotube (CNTs) to form coaxially layered CNTs/MoS2-X, which was then synthesized with highly dispersed titanium dioxide nanoparticles (TiO2) to synthesize CNTs/MoS2-X/TiO2 composites for the detection of human sweat H2O2 and phosphorylated proteins, respectively. This structure, with its sulfur vacancies and coaxial layering, significantly improved sensitivity of electrochemical sensors, allowing it to detect H2O2 in a range of 0.01–1 mM with a detection limit of 4.80 μM, and phosphoproteins in a range of 0.01–1 mg/mL with a threshold of 0.917 μg/mL. Furthermore, the miniature sensor demonstrates outstanding performance in detecting analytes in both simulated and real sweat. Comprehensive biosafety assessments have validated the compatibility of the electrode material, underscoring the potential of sensor as a reliable and non-invasive method for tracking biomarkers linked to CNS disorders. This microfluidic wearable electrochemical biosensor with high performance and biosafety features shows great promise for the development of cutting-edge wearable technology devices for tracking CNS disease indicators.