The ability to accurately analyze perfluoroalkyl substance (PFAS) levels in beef is imperative in order to effectively assess food-safety risks and ensure consumer safety because PFASs are harmful and prevalent in beef. In this study, we developed a rapid and accurate method for the simultaneously determination of the 17 PFASs in beef using dispersive solid-phase extraction (d-SPE) and ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS), and optimized the mobile phase system, extraction solvent, and d-SPE materials. Samples were finally extracted using 0.1% (v/v) formic acid in acetonitrile, cleaned using d-SPE with PSA, C18, GCB, and EMR-Lipid, separated using an Acquity Premier BEH C18 column (100 mm×2.1 mm, 1.7 μm) with 0.5 mmol/L ammonium fluoride aqueous solution and methanol as the mobile phases at a flow rate of 0.3 mL/min. Analytes were detected in negative ion switching mode (ESI-) with multiple reaction monitoring (MRM) scanning, and quantitatively analyzed using the internal standard method. The 17 PFASs exhibited linearity in the 0.2-20.0 μg/L range under the optimal experimental conditions, with correlation coefficients of 0.9915-0.9999. The method delivered limits of detection (LODs) of 0.003-0.007 μg/kg and limits of quantification (LOQs) of 0.01-0.02 μg/kg. The 17 PFASs exhibited recoveries of 71.1%-127.4% with RSDs of 0.6%-14.4% when spiked at three levels (0.05, 0.5, and 1.8 μg/kg). We optimized the mobile phase system, which revealed that, compared with 2.0, 5.0, and 10.0 mmol/L ammonium formate or ammonium acetate in aqueous methanol, 0.5 mmol/L ammonium fluoride in aqueous methanol exhibited higher sensitivities for all the 17 PFASs, with PFASs bearing long-chain carboxylic acids (C10-C18) showing 1-2 fold increases in sensitivity. PFASs do not dissociate in acidic environments, favoring their entry into the organic phase. Therefore, we investigated the effect of extractant acidity, which revealed that the 17 PFASs were better extracted using 0.1% (v/v) formic acid in acetonitrile. The beef matrix has a complex composition; consequently, d-SPE adsorbents were required to purify samples and reduce matrix effects. The purification effects of four adsorbents (PSA, C18, GCB, and EMR-Lipid) toward the 17 PFASs and the amount of EMR-Lipid used were investigated, which revealed that 100 mg PSA+80 mg C18+40 mg GCB+150 mg EMR-Lipid exhibited superior matrix-purification behavior. We also investigated the effects of various injection solutions and types of syringe filter, with pure methanol selected for reconstitution and high-speed supernatant centrifugation applied prior to injection. The developed method is simple, rapid, sensitive, and reproducible, and can be used to simultaneously, rapidly, and accurately determine various perfluoroalkyl compounds in beef.