Vitamin K₁ is one of the important hydrophobic vitamins in fat-containing foods. Traditionally, lipase is employed in the determination of vitamin K₁ to remove the lipids, which makes the detection complex, time-consuming, and insensitive. In this study, the determination of vitamin K₁ in fat-containing foods was developed based on ultrasound-assisted extraction (UAE), solid-phase extraction (SPE) combined with liquid chromatography–tandem mass spectrometry (LC-MS/MS). The optimal conditions for extraction of vitamin K₁ were material–liquid ratio of 1:70 (g/mL), extraction temperature of 50 °C, extraction power of 700 W, extraction time of 50 min, material-wash fluid ratio of 1:60 (g/mL), and 8 mL of hexane/anhydrous ether (97:3, v/v) as the elution solvent. Then, vitamin K₁ was analyzed on a ZORBAX SB-C18 column (50 mm × 2.1 mm, 1.8 μm) by gradient elution with water (0.01% formic acid) and methanol (0.01 formic acid + 2.5 mmol/L ammonium formate) as the mobile phase. The limit of detection (LOD) and limit of quantification (LOQ) were 0.05 and 0.16 μg/kg, respectively. Calibration curve was linear over the range of 10–500 ng/mL (R² > 0.9988). The recoveries at three spiked levels were between 80.9% and 119.1%. The validation and application indicated that the proposed method was simple and sensitive in determination of vitamin K₁ in fat-containing foods.