This research aims to study the conservation of carbon source in denitrification process by external carbon source step-feed technique. The main objective is to find the optimum amount of carbon source for denitrification process.In this experiment, an anaerobic fluidized bed reactor with non-internal recirculation using rubber granule as a media was conducted. A fixed nitrate concentration as 100 mg/L was added into the synthetic wastewater and various COD concentration were prepared from glucose as carbon source. The study consisted of 4 experimental steps. The first step is the reactor start-up. According to stoichiometry, a COD/NO3- -N ratio was fixed as 5:1 to promote active biofilm in the reactor. After steady state, the result showed that nitrate and COD removal efficiencies were 97.59±8.30% and 85.50±9.07%, respectively. In the second step, a COD/NO3- -N ratio was decreased to 1:1 to represent a low COD contained wastewater. The results indicated the reduction of nitrate and COD removal efficiencies as 25.93±9.89% and 80.36±9.23%, respectively. In this step, the effluent from various sampling ports along the reactor height were collected and analyzed. Nitrate and COD concentration were evaluated to indicate the point of insufficient carbon source. The result presented that almost COD was rapidly reduced at the bottom of the reactor. Therefore, in the third step, external carbon source was fed (as step-feed) at the sampling port number 2. The result showed that the highest nitrate removal efficiency occurred at the COD/NO3- -N ratio of 3.6:1 as 96.86±3.65%. Moreover, the COD removal efficiency was 95.74±1.84%. In the last step, the microbial population was study via PCR-DGGE technique. The results illustrated that denitrifying bacteria population did not change from the effect of organic carbon source. The result clearly revealed that difference quantity of carbon source in anaerobic fluidized bed reactor had not affected to structural of denitrifying bacteria. It can be concluded that the only one point of carbon source adding at a suitable position of the anaerobic fluidized bed reactor with non-internal recirculation using rubber granule as a media can achieve high nitrate reduction performance and the effluent contained very low nitrate and COD concentration.