As a natural fluorescent material, the practical application of lignin fluorescence was hindered due to the low fluorescence quantum yield (QY). Inspired by its aggregation fluorescence behavior, the effect of microstructure-scale on lignin fluorescence was studied from two levels, the molecular weight and colloidal sphere. It was demonstrated that with the decrease of lignin microstructure-scale, the non-radiative dissipation and reabsorption effect of lignin fluorescence would be weak, resulting in high emission efficiency. On this basis, hydrogenolysis was used to obtain small molecular fragments and reduce content of reabsorbing groups of lignin, of which the QY was greatly increased by 35 times to about 12%. In addition, the emission peak of lignin was the fluorescence addition of its main structural units. The long-wavelength emission peak was often the illusion from the reabsorption effect but not duo to the formation of conjugated structure. This work provided a potential method for the preparation of high QY lignin and an in-depth understanding of lignin fluorescence.