[Objective] Acanthamoeba spp. and Hartmannella vermiformis are free-living amoebae (FLA), which are widely ubiquitous in soils and various aquatic environments. Certain species in these two genus were recognized as potential opportunistic pathogenicity for human and animals. It has practical significance of developing a highly specific, sensitive and reproducible assay which based on real-time fluorescence quantitative PCR for identifying amoebae. [Methods] we employed a culture-independent method, the real-time quantitative PCR assays to detect 18S rRNA genes of Acanthamoeba spp. and H. vermiformis using BRYT Green? dye in different processes of rural domestic wastewater treatment plant. [Results] The results indicated that the most frequently amoebae discovered were Acanthamoeba spp. and H. vermiformis which appear in all treatment system stages. The concentrations of two amoebae genus were up to 8.70×105 and 1.84×106 copies/L in untreated wastewater, respectively. Compared with untreated wastewater, the abundance of amoebae in adjusting tank, aerobic tank and membrane tank decreased in one or two orders of magnitude, while Acanthamoeba spp. was one order of magnitude higher in treated wastewater than untreated wastewater. [Conclusion] Hence, it should be paid much attention to amoebae having health risk and it is necessary to use amoebae as an additional reference standard for domestic wastewater treatment.