This paper, for the first time, reports the optimization of the critical medium components for bio-surfactant production from achromobacter xylos strain GSR21 using statistical experimental design. Response surface methodology (RSM) was employed to determine the optimal levels of process variables (agar powder, yeast extract, FeSO₄7H₂O, and KH₂PO₄). Central composite design (CCD) of RSM was used to study the four variables at five levels, and bio-surfactant concentration was measured as response. Regression coefficients were calculated by regression analysis, and the model equation was determined. R² value for bio-surfactant (g/L) was tested to be 0.7222, indicating that the model fitted well with the experimental results. Verification of the mathematical model was conducted by performing the experiment with the predicted optimized values, and bio-surfactant yield was found to be 9.69 g/L. Validation of the predicted model was fitted 96.9% with the experimental results conducted under the optimum conditions. Agar powder and yeast extract was identified as efficient components for bio-surfactant (achromobacter xylos GSR21) production.