Wearable biosensors have great advantages and application potential in the field of real-time detection. However, it is still a challenge to construct an electrically conductive and mechanically flexible sensing interface as the working electrode in an electrochemical sensor. In this work, a green and efficient strategy is proposed to optimize the chemical structure of precursor materials for the preparation of a flexible biosensor. The introduction of phosphated lignin effectively increases the molecular interaction in the electrospinning system, improving the micro-morphology, flexibility, and thermal stability of biomass-based carbon nanofibers (CNFs). The rich active sites and high graphitization degree provide abundant access to uric acid molecules and accelerate electron transmission. Benefiting from these compelling features, the fabricated wearable biosensor can accurately and selectively detect uric acid in artificial urine. This work offers a promising approach for the fabrication of wearable biosensors and has a broad application prospect in personalized diagnostic and miniaturized power device fields.