In the food industry, bacterial cells usually adhere to equipment surfaces, forming biofilms that may cause persistent contamination. This study aimed to identify the key genes responsible for the stronger biofilm-forming capability of the Listeria monocytogenes LMB 33426 strain compared to that of the L. monocytogenes CICC 21662 strain through comparative genomics. Additionally, the expression of genes and related metabolic pathways of LMB 33426 and CICC 21662 strains were analyzed at the transcriptional level by high-throughput sequencing technology to uncover key differentially expressed genes between planktonic and biofilm cells of those two strains. Subsequently, the key genes found to present differences that were uncovered by those genome-wide and transcriptomic analyses were used to construct gene deletion strains. The crystalline violet assay and motility assay showed that GL002291, GL002712 and lmo1438 genes were involved in the regulation of biofilm formation as well as motility. The hydrophobicity and auto-aggregation ability assay results demonstrated an association between the clpB, lmo1438, and lmo0294 genes and bacterial adhesion. However, no significant differences were found regarding this association in the GL002291 and GL002712 genes. This study elucidates some potential regulatory genes associated with biofilm formation in L. monocytogenes, and laying a theoretical foundation for future research.