To supply the valuable operating parameters for the popular usage of the new denitrifying phosphors removal process, it is essential to study the dominant biochemical reactions and the characteristics of denitrifying phosphorus removing bacteria (DPB). Thus, parallel batch experiments using DPB sludge were carried out to assess the effect of substrates (sewage, HAc, and endogenous carbon source) on denitrifying dephosphorus removal efficiency in this study. The results showed that the initial specific phosphorus release rate increased with the high concentration of the short-chain volatile fatty acids ratio in the influent, and sufficient phosphorus was released by DPB. This improved the subsequent denitrification and phosphorus uptake efficiency. The specific endogenous denitrification mainly relies on the internal carbon source (PHB) stored by poly-P bacteria. Denitrifying phosphorus removing bacteria were very hungry when the internal PHB was consumed. Consequently, the specific endogenous denitrification rate was low and the phosphorus uptake did not happen. On the other hand, in the experiment, the denitrifying phosphorus removal performance under two temperature conditions (8 10?C and 25 26?C) was also investigated and analyzed. It was found that the lower temperature decreased the specific phosphorus release and uptake rate, but did not inhibit the denitrifying phosphorus removal completely. Therefore, the negative influence of the low temperature on the overall phosphorus removal was not significant.