It is highly desirable to develop a Bacillus thuringiensis (Bt) formulation with high UV resistibility to increase its application duration in the field. In this work, we studied spent mushroom substrate extract (SMSE) for its anti-UV activity on Bt. The results demonstrated that SMSE could efficiently protect the active components of Bt from UV irradiation. After 96 h of UV exposure, the residual toxicity of Bt fermentation of the SMSE (0.6%, w/v) treatment was 17 times higher than that of the control treatment. SMSE was mainly composed of lignin (77.69%), carbohydrate (7.58%) and protein (4.18%). The anti-UV activity was shown by the lignin and carbohydrate fractions, but not the protein fractions. In addition, it was found that lignin and lignin-carbohydrate complex (LCC) in the SMSE were a type of lignin with low sugar content, low thermal stability, narrow molecular weight distribution and were typical gramineous plant lignin. Lignin and LCC possessed highly UV-screening effects and reactive oxygen species scavenging ability that could improve the UV stability of Bt formulation. Our study provides a green method for the utilization of SMS biowaste and increased the UV stability of Bt formulation.