The biodegradation process was investigated using an isolated single bacterial culture of P. putida. The P. putida was able to reduced 75-80% pollution load within 48 hrs under aerobic condition as term of Chemical Oxygen Demand (COD) in River Water containing Phenol (RWP)). Obtained data showed that P. putida was potent bacterial culture for RWP treatment. The influence of three process parameters such as temperature (20-500C), pH (4-9) and agitation speed (30-150 rpm) influenced the rate of biodegradation extent. Central composite design (CCD), an experimental design for response surface methodology (RSM), was used to create a set of 36 experimental runs needed for optimizing operating conditions. The optimal degradation conditions were determined by response surface methodology based on three-variable central composite design to obtain maximum reduction and to determine the significance and interaction effect of the variables on degradation. The optimal conditions of response were found to be 35 0C, pH 8.05 and 120 rpm agitation speed, respectively, giving an experimental degradation value of 80.07%. Very high regression coefficient between the variables and the response (R2 = 0.6447) indicated a good evaluation of experimental data by polynomial regression model.