Abstract: In response to the environmental and health risks posed by antibiotic residues in soil, as well as the challenges of existing detection methods, which include low sensitivity, weak anti-interference ability, and inefficient sample preparation when simultaneously analyzing multiple classes of antibiotics, an ultra-high-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was established for the simultaneous detection of 72 antibiotics in soil. This method encompasses seven major classes of compounds: sulfonamides, quinolones, tetracyclines, macrolides, chloramphenicol, β-lactams, and others. By systematically optimizing the mass spectrometry parameters and chromatographic conditions, the method enhances detection sensitivity and accuracy through positive and negative ion switching scans and simultaneous multiple reaction monitoring (sMRM) mode. In the sample preparation process, a novel extraction agent comprising acetonitrile-EDTA-McIlvaine buffer was utilized. After three extraction steps involving ultrasound, oscillation, and centrifugation, method validation results indicated that the target antibiotics exhibited good linearity within the range of 5 to 300μg/L (R²>0.99), with detection limits ranging from 0.06 to 0.57μg/kg and spiked recovery rates between 51.0% and 110.4%, along with relative standard deviations of 1.24% to 7.76%. This method was successfully applied to the detection of actual urban soil samples, identifying a total of 24 antibiotics across six major classes, predominantly consisting of quinolones and sulfonamides, with most recovery rates exceeding 70%. This study presents a reliable analytical method that outperforms existing technologies in terms of simultaneous detection of compound varieties, sample preparation efficiency, and method applicability, thereby providing a robust analysis for monitoring soil antibiotic pollution and assessing associated risks.