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Antibiotics and hormones widely exist in fertilizers and manures, which are excessively used in agriculture and animal husbandry. Considering their potential harm to the environment and human health, the detection of antibiotics and hormones has become a necessity. However, current methods find it difficult to simultaneously extract and detect antibiotics and hormones in soil and to maintain a high level of accuracy and a low cost. In this study, a straightforward, convenient, and simultaneous extraction and detection method of a representative antibiotic (sulfamethoxazole, SMZ) and hormone (17β-Estradiol, E2) in soil has been established. Ultrasound-assisted extraction (UAE) was used in the pretreatment process and high-performance liquid chromatography with the ultraviolet detector (HPLC-UV) method was then chosen in the detection process. By means of single factors and response surface experiments, optimal extraction conditions were a 41-mL buffer solution (pH 4.27) mixed with 1 g of soil sample, an ultrasonication time of 36 min, an ultrasonication temperature of 25 °C, and two extraction cycles. The detection limits of 0.3−10 μg/kg and quantification limits of 1−30 μg/kg have been obtained. Finally, the optimized simultaneous extraction and detection method was validated by three different real soil samples with recoveries ranging from 79.49% to 86.47%.