Matrix solid-phase dispersion extraction was applied to the extraction of sulfadiazine, sulfamerazine, and sulfamethazine from human and animal bloods. The separation and determination of the analytes were carried out by high-performance liquid chromatography. The effects of the types of the dispersion adsorbents and elution solvents were investigated, and the highest recovery was obtained when diatomaceous earth was used as the dispersion adsorbent, while acetone was used as the elution solvent. Under the optimal conditions, the linear range for determining the sulfonamides in blood samples was 0.020-10.0 µg/mL, and the average recoveries of the three sulfonamides were higher than 87.5%.