Coix seed is an important food and traditional Chinese medicine in China and other Asian countries. Notably, coix seed is currently being used as a traditional medicine for the treatment of COVID-19 in China. However, coix seeds are generally contaminated by mycotoxins, and this risk cannot be ignored. In this paper, we developed a method that involves direct extraction and UHPLC-HRMS analysis for the simultaneous detection of 24 mycotoxins in coix seeds. UHPLC-HRMS instrument and data acquisition parameters, and the sample pretreatment were optimised. One-step extraction showed several advantages compared to the three commercial solid-phase extraction clean-up methods, including ease of use, reduced time of sample preparation, low cost, good recovery, and acceptable matrix effect. The method validation results indicate that all mycotoxins have good linearity and sensitivity. Recoveries were between 74.2–101.1%, and RSD ranged from 0.1–5.8%. The LOQs for 24 mycotoxins were in the range of 0.5–100 µg/kg. To survey the contamination levels of these mycotoxins in commercial coix seeds, more than 70 samples were collected from Chinese markets and were analysed using the newly developed method. Zearalenone (positive ratio: 98.7%, range:1.1–1562 µg/kg), deoxynivalenol (positive ratio: 87%, range: 8.4–382.5 µg/kg), nivalenol (positive ratio: 85.7%, range: 26.8–828.2 µg/kg), fumonisin B₁ (positive ratio: 84.4%, range:2.5–314.5 µg/kg), fumonisin B₂ (positive ratio: 75.3%, range:1.6–72.8 µg/kg), fumonisin B₃ (positive ratio: 48%, range:1.0–203.6 µg/kg), aflatoxin B₁ (positive ratio: 29.9%, range: 0.39–14.7 µg/kg), sterigmatocystin (positive ratio: 29.9%, range: 1.4–51.6 µg/kg), and tenuazonic acid (positive ratio: 19.5%, range 36.1–105.7 µg/kg) were the most frequent mycotoxin contaminants. These results highlight the importance of routine monitoring and control of mycotoxins in coix seeds.