Long-term or inappropriate use of glyphosate may damage the non-target organisms and environment. Microbes are important biological resources for bioremediation. In recent years, the method of using microbes and its catabolic enzymes to remediate organophosphorus pesticides in the environment has shown a promising potential in application. It is the main direction of research on organophosphorus pesticide degradation. This study aimed to investigate the degradation of glyphosate by an efficient phosphate solubilizing bacterium Burkholderia. multivorans WS-FJ9 which was isolated from pine rhizosphere. Strain WS-FJ9 was grown on the plates of nutrient agar (NA) with different concentrations of glyphosate to determine the tolerance of strain WS-F19 to glyphosate. Glyphosate can be used by strain WS-FJ9 as the sole sources of carbon, nitrogen and phosphorus, respectively. The degradation dynamical parameters of strain WS-FJ9 on glyphosate were determined using low flooding quantity batch reactor method (FBR). Plackett-Burman (PB) design, central composite design (CCD), and Response Surface Methodology (RSM) were employed to screen and optimize the main factors of strain WS-FJ9 degrading glyphosate. The maximum concentration of tolerance to glyphosate for strain WS-FJ9 to remain efficiently degrading glyphosate was 0.4%. The affinity constant (K_s) of strain WS-FJ9 to glyphosate was 65 μL/mL and the minimal concentration (S_min) of glyphosate degraded by WS-FJ9 was 21.9 μL/mL. Three key factors (cultural temperature,glucose and ammonium sulfate) for the glyphosate degradation by strain WS-FJ9 were selected using PB design. The quadratic model for the three significant factors was established using CCD design and RSM with glyphosate degradation rate as the target response. Under the optimal degradation conditions, the incubation temperature was 27.7℃, and the amount of glucose and ammonium sulfate supplemented were 9.67 g/L and 0.50 g/L, respectively. The degradation rate of glyphosate by strain WS-FJ9 reached 72.83%.