Comprehensive screening of harmful chemical drugs in food is an essential analytical tool in the investigation of food poisoning. Meanwhile, determining harmful chemical drugs in biological matrices is a supplement to support the screening results in food. In this study, we developed a screening method for 315 harmful chemical drugs in food and biological matrices based on ultrahigh-performance liquid chromatography quadrupole time of flight mass spectrometry. The food and gastric juice sample was extracted by 1.0% formic acid acetonitrile and acetonitrile, respectively, and cleaned up by a C18 sorbent. Urine and serum were directly extracted by 1.0 % formic acid acetonitrile. Then the data was acquired and screened for drugs using a self-built library. The recovery, accuracy, and limit of quantitation for methodology were investigated. The method was validated by other three laboratories in different areas in China using different mass spectrometric techniques. For screening, the coefficient of variation of the retention times was below 5%. The recoveries for the drugs in nine matrices were 70-120%.At last, the developed method was applied to a real incident, demonstrating its suitability for the sensitive and fast screening of harmful chemical drugs in food and biological samples.