Burkholderia pyrrocinia JK-SH007 is an important biocontrol strain for the prevention and treatment of poplar canker disease. Its powerful biocontrol function is inseparable from its successful colonization of poplar trees. Bacterial biofilms can ensure the long-term colonization of a host. To explore the mechanism of action of biofilms in the biocontrol process, we manipulated various exogenous factors to explore the morphology of the JK-SH007 biofilm in vitro. The addition of glycerol and MgSO4 to TSB medium stimulated biofilm production, increased the resistance of JK-SH007 to disease, enhanced the survival of JK-SH007 in nutrient-poor environments and maintained the antagonistic ability of JK-SH007 against the poplar canker pathogen. Therefore, we constructed and optimized a biofilm-forming system to produce a large number of stable JK-SH007 biofilms. The optimized system showed that the optimal incubation time for JK-SH007 biofilm formation was 14 h, the optimal temperature of the static culture was 25 °C, and the optimal pH was 5. The optimal medium for biofilm formation was TSB medium, 1% glycerol and 50 mM MgSO4. RT-qPCR experiments showed that an increase in the expression of the suhB gene promoted JK-SH007 biofilm formation, while an increase in the expression level of the ropN gene inhibited JK-SH007 biofilm formation. The possible mechanism by which JK-SH007 was inhibited by biofilm formation under natural culture was revealed. These results indicate the importance of adding nutrients to JK-SH007 biocides produced on a commercial scale. This is the first report of JK-SH007 producing a long-lasting biofilm that guarantees antagonism.