Abstract: Berberine bridge enzyme（BBL） plays a key catalytic role in the last step of nicotinoid biosynthesis. In order to study the function of tobacco BBLc gene of BBL gene family, the CRISPR/Cas 9 gene editing technology were used to construct BBLc knockout vector. 35 resistance seedlings were screened through kanamycin with genetic transformation of tobacco by Agrobaterium-mediated method and 7 mutant seedlings were identified by PCR and RT-qPCR technology. The degree of leaf fold on mutant seedlings was higher than that of wild types, and the other agronomic traits on mutant seedlings were no obvious difference from those of wild types. The nicotine content detected by HPLC of the mutant leaves was decreased 24.2% below average, and the content of mutant CB 10 plant was 0.88%, which had the maximum drop by 37.6%. The resutls showed that the decrease of nicotine content was caused by decrease of BBLc gene expression.