Aflatoxin B1 (AFB1), a pollutant of agricultural products, has attracted considerable attention in recent years, due to its potential impact on health. In the present study, Bacillus licheniformis (BL010) was demonstrated to efficiently degrade AFB1, reducing over 89.1% of the toxin content within 120 h. A crude enzyme solution of BL010 exhibited the highest degradation level (97.3%) after three induction periods. However, uninduced BL010 bacteria was not capable of reducing AFB1. Furthermore, high performance liquid chromatography (HPLC) analysis showed that while a cell-free extract caused a significant decrease in AFB1 content (93.6%, p < 0.05), cell culture fluid treatment did not significantly degrade AFB1. The biotransformation products of AFB1 were detected and further identified by quadrupole time-of-flight liquid chromatography–mass spectrometry (LC-Q-TOF/MS); these corresponded to a molecular formula of C12H14O4. A sequence analysis of whole BL010 genes with a bioinformatics approach identified the secondary structures of two degrading enzymes (Chia010 and Lac010), providing an important basis for subsequent homology modeling and functional predictions.