To study the lipopolysaccharide (LPS) of Aeromonas hydrophila, LPS was extracted by hot phenol-water method, purified by enzymatic hydrolysis and chloroform/methanol precipitation. The yield of LPS and the contents of protein, DNA were determined. The purity of LPS was analyzed by SDS-PAGE and silver staining．Ana-1 cell model was established to evaluate the toxicity and biological activity of LPS samples. The results showed that the yield of LPS was 1.22%, and the contents of protein and nucleic acid were 1.53% and 0.79% respectively. The main bands of SDS-PAGE were clear and located at same position as the LPS standard. After LPS treatment, the viability of Ana-1 cells decreased by 21.20%, and the fluorescence intensity of Ca²⁺ was 1.67±0.19, without significant difference compared with LPS standard. The LPS samples extracted from Aeromonas hydrophila had high purity, toxicity and biological activity. This study provided technical support for the development of subsequent extraction and detection methods.