Objective Screening bio-samples from a food poisoning event based on ultra-high-performance liquid chromatography quadrupole time of flight mass spectrometry (UPLC-QTOF MS), and to provide strategies and references for patient treatment as well as the event response. Methods Vomits, gastric lavage and food samples were extracted by acidic acetonitrile and purified by C18 sorbent and analyzed using UPLC-QTOF MS under data independent acquisition mode. Peak identification and spectrum library retrieval were carried out by the Unifi software. Results Hyoscyamine was detected in 5 human samples and the concentration was below 1 mg/kg. Azithromycin was also screened in one gastric lavage sample. No metabolites of hyoscyamine was found, which was consistent with the result of rat exposure experiment. Conclusion According to the screened result, it was inferred that this event was a family food poisoning caused by hyoscyamine. The UPLC-QTOF MS screening is of great significance in dealing with food poisoning emergencies.