Enhanced biological phosphorus removal (EBPR) is a commonly used and sustainable method for phosphorus removal from wastewater. Poly-β-hydroxybutyrate (PHB), polyphosphate, and glycogen are three kinds of intracellular storage polymers in phosphorus accumulation organisms. The variation of these polymers under different conditions has an apparent influence on anaerobic phosphorus release, which is very important for controlling the performance of EBPR. To obtain the mechanism and kinetic character of anaerobic phosphorus release, a series of batch experiments were performed using the excessively aerated sludge from the aerobic unit of the biological phosphorus removal system in this study. The results showed that the volatile suspended solid (VSS) had an increasing trend, while the mixed liquid suspended sludge (MLSS) and ashes were reduced during the anaerobic phosphorus release process. The interruption of anaerobic HAc-uptake and phosphorus-release occurs when the glycogen in the phosphorus-accumulating-organisms is exhausted. Under the condition of lower initial HAc-COD, HAc became the limiting factor after some time for anaerobic HAc uptake. Under the condition of higher initial HAc-COD, HAc uptake was stopped because of the depletion of glycogen in the microorganisms. The mean ratio of ΔρP/Δρ, Δρ/ΔρPHB, ΔρP/ΔCOD, and ΔρPHB/ΔCOD was 0.48, 0.50, 0.44, and 0.92, respectively, which was nearly the same as the theoretical value. The calibrated kinetic parameters of the HAc-uptake and phosphorus-release model were evaluated as follows: Q was 164 mg/(g °h), Q was 69.9 mg/(g °h), K was 0.005, and KCOD was 3 mg/L. An apparently linear correlation was observed between the ratio of ΔρP/ΔCOD and pH of the solution, and the equation between them was obtained in this study.