Biodegradation of Phenanthrene (PHE) was studied in aqueous culture to demonstrate the potential of the mixed culture in degrading high concentration of PHE. The experiments were conducted to monitor biodegradation of Phenanthrene for duration of 6 days. Biodegradation of PHE was successfully achieved in low and middle concentration by the isolated mixed culture. A full factorial Central Composite Design of experiments was used to construct response surfaces with the removal, the extent of PHE degradation and the specific growth rate as responses. The initial Phenanthrene concentration (X1) and the reaction time (X2) were used as design factors. The result was shown that experimental data fitted with the polynomial model. Analysis of variance showed a high coefficient of determination value in the range of 0.936–0.999. The maximum biodegradation of PHE in terms of the removal of PHE (Y1) was found to be 0.100 mg/mg (degraded PHE/initial PHE). It was observed that optimum biodegradation of PHE can be successfully predicted by RSM.