Bifidobacteriaare representative intestinal probiotics that have extremely high application value in the food and medical fields. However, the lack of molecular biology tools limits the research on functional genes and mechanisms ofbifidobacteria. The application of an accurate and efficient CRISPR system to genome engineering can fill the gap in efficient genetic tools forbifidobacteria. In this study, CRISPR system ofB. animalisAR668 was established, which successfully knocked outgene 0348andgene 0208. The influence of different homology arms and fragments on the knockout effect of the system was explored. In addition, the inducible plasmid curing system ofbifidobacteriawas innovatively established. This study contributes to the genetic modification and functional mechanism analysis ofbifidobacteria.