The invention relates to a method for detecting the enantiomer of ofloxacin in seawater, which comprises the following steps: vacuum filter the seawater, then adjust the pH to 2-4, and vacuum filter again to obtain pretreated seawater; Phase extraction column, seawater passes through the column, after the extraction is completed, rinse with deionized water, vacuum dry, and elute in 2-5 times, and the eluent is dried in a nitrogen blowing instrument in a water bath at 30-35°C , the residue was redissolved with ethanol, micropore nanofiltration, and the filtrate was used for later use; the concentration of the enantiomer of ofloxacin was detected by high performance liquid chromatography. The present invention optimizes the solid-phase extraction conditions, so that the time of solid-phase extraction is shortened and the efficiency becomes high, while the high-performance liquid chromatography method for analyzing the concentration of ofloxacin enantiomers has high precision, fast speed, simple operation and low cost. Low; the detection method has a good linear relationship to the target compound, high recovery rate, small relative deviation, low toxicity of the reagents used, less usage, and is environmentally friendly.