Bacterial conjugation constitutes a major horizontal gene transfer mechanism for the dissemination of antibiotic-resistant genes (ARGs) among human pathogens. The spread of ARGs can be halted or diminished by interfering with the conjugation process. In this study, we explored the possibility of using an rbsB gene as a single target to inhibit plasmid-mediated horizontal gene transfer in Escherichia coli by CRISPR interference (CRISPRi) system. Three single-guide RNAs (sgRNAs) were designed to target the rbsB gene. The transcriptional levels of the rbsB gene, the conjugation-related genes, and the conjugation efficiency in the CRISPRi strain were tested. We further explored the effect of the repressed expression of the rbsB gene on the quorum sensing (QS) system and biofilm formation. The results showed that the constructed CRISPRi system was effective in repressing the transcriptional level of the rbsB gene at a rate of 66.4%. The repressed expression of the rbsB gene resulted in the reduced conjugation rate of RP4 plasmid by 88.7%, which significantly inhibited the expression of the conjugation-related genes (trbBp, trfAp, traF and traJ) and increased the global regulator genes (korA, korB and trbA). The repressed rbsB gene expression reduced the depletion of autoinducer 2 signals (AI-2) by 12.8% and biofilm formation by a rate of 68.2%. The results of this study indicated the rbsB gene could be used as a universal target for the inhibition of conjugation. The constructed conjugative CRISPRi system has the potential to be used in ARG high-risk areas.