[Background] Pseudomonas chlororaphis HT66 is a plant growth-promoting rhizobacterium (PGPR) with biocontrol safety and high yield of phenazine-1-carboxamide (PCN). It has broad application prospects in biological control, ecological agriculture and sustainable agriculture. SuhB, a small non-coding RNA, involved in the metabolic regulation of multiple processes in cells. [Objective] This study explored the effect of suhB gene on the biocontrol ability of in P. chlororaphis HT66. [Methods] we constructed the suhB deletion mutant HT66ΔsuhB by homologous recombination method and the suhB complemented mutant HT66ΔsuhB-pBBR-suhB by plasmid. They were used to explore the effect of suhB gene on strain growth, biofilm formation, swarming motility and PCN synthesis. [Results] The mutant HT66ΔsuhB grew slowly, the plateau period delayed by 12 h and its biomass decreased to 61.6% of the wild type. The maximum yield of mutant strain in KMB medium can reach 109.5 mg/g (per DCW), which was 2.1 times that of wild type. The biofilm formation increased significantly, which was 1.8 times that of the wild type. However, the swarming motility of HT66ΔsuhB was defective. On the swarm plate, the movement radius of the wild strain was 21 mm, while the movement radius of the mutant strain was 9.7 mm. The suhB gene complemented mutant is similar to wild type in the above aspects. Compared with the wild type, there is no difference on the expression of phzI at the overall level in mutant HT66ΔsuhB. On the contrary, the expression of phzR increased significantly, which is 3.1 times that of the wild type. In addition, the expression of phzAp at the transcriptional level is 1.8 times that of the wild type. [Conclusion] The regulation of suhB gene in P. chlororaphis HT66 participates in the growth, biofilm formation, swarming motility and PCN synthesis. This research provides a theoretical basis for the metabolic transformation and biocontrol applications of P. chlororaphis HT66.