In a biological treatment process, hydraulic retention time (HRT) has a certain effect on the operation of the reactor. This study investigated the effect of HRT on carbon sequestration in a two-stage anaerobic ammonium oxidation (anammox) process using a partial nitrification reactor and anammox reactor to determine the optimal carbon sequestration operating conditions. Molecular biotechnology was used to analyze the sludge in the reactor in order to explore the denitrification performance and to determine the carbon sequestration pathway of the microorganisms. The results show that the partial nitrification stage had the highest carbon sequestration rate (0.319 mg/mg·N) when the nitrogen loading rate (NLR) was 0.44 kg·N/m3/d. The NLR of the anammox stage was 0.13 kg·N/m3/d. When the HRT was 33.4 h, the carbon sequestration of the anammox reaction was at its highest, reaching 0.183 mg/mg·N. The results of microbial analysis show that the carbon-fixing gene cbbLR1 was present in the sludge samples during the anammox and partial nitrification stages, and that there was a Calvin cycle carbon sequestration pathway during the growth process. However, the existence of a gene for reducing and immobilizing CO2 by the acetyl-CoA pathway was not detected; further research is thus needed.