Abstract: Environmental DNA (eDNA) technology has been widely used in the study of aquatic biodiversity in recent years. In this study, different filter membrane, storage method of membrane and DNA extract kits were used to evaluate effectiveness of eDNA enrichment from the brackish water in the Pearl River Estuary. Four kinds of filter membranes with diameter of 47 mm and pore size of 0.45 μm (Nitrocellulose membrane, cellulose acetate membrane, glass fiber membrane and polycarbonate membrane) were selected. Four kinds of membrane preservation methods were adopted and two kinds of DNA extract kits were used. The results show that filter membrane materials and preservation methods had a significant impact on the yield of eDNA. The highest concentration of eDNA was obtained by cellulose acetate membrane. Under the condition of the same quality of DNA yield, the concentration of eDNA extracted by Marine Animal Tissue Genomic DNA Kit was higher than that by DNeasy Blood and Tissue Kit. The concentration of eDNA obtained from ‘liquid nitrogen’ storage method was the highest among the four storage conditions. However, the concentration of eDNA was acceptable for alcohol preserved method at room temperature when cryopreservation was not available. Filtering water samples immediately after sampling can prevent eDNA degradation effectively. This study established a systematic protocol for eDNA extraction of water samples from the brackish ecosystem in the Pearl River Estuary, providing references for eDNA research in similar waters.