Many studies are still limited to focusing on the primary structure of biomass macromolecules, ignoring the impact of the multi-level structure of macromolecules and their conformational evolution on the high-value utilization of biomass. In this paper, a novel and clever strategy is designed and executed to regulate and immobilize the multi-level structure of lignin macromolecules. Lignin with different spatial conformations exhibits differences in physical properties and processability, despite their chemical primary structures being highly similar. In order to clearly and intuitively study the impact of multi-level structures, lignin-based carbon nanofibers (CFs) with high requirements for processing and precursor properties have been selected as the high-value utilization. The introduction of lignin with optimized multi-level structures effectively improves fiber morphology, uniform diameter, and flexibility. The resulting lignin-based CFs exhibit excellent refractoriness, stability, flame sensitivity (response time of only 1 s), and good energy storage properties (specific capacitance up to 226.1F/g). Furthermore, the CFs moist-electric generator has a high power generation efficiency, and the output voltage of a single device reaches 0.649 V and the output current is up to 160 μA. This work may explain a long-standing issue: why do lignin macromolecules with similar primary structures exhibit many differences in physical and processing properties, which is of great significance for promoting the industrial utilization of lignin macromolecules.