Abstract: A method for rapid screening of 32 hormone residues in animal tissues by liquid chromatography quadrupole time of flight mass spectrometry (LC-Q-TOF/MS) was established. Samples were extracted by acetonitrile and ethyl acetate and extracted was purified by enhanced lipid removal adsorbent (EMR). After salting out, extract were further purified by PSA and functionalized polystyrene/two vinyl benzene (PEP-2), with 0.1% formic acid water-0.1% formic acid was used as mobile phase for gradient elution, in Eclipse plus-C₁₈ (3.0 mm×100 mm, 1.8 μm) column on the chromatographic column separation. The mass spectrum data were collected in Q-TOF/MS positive ion full scan mode. The retention time, accurate molecular mass number, isotopic abundance ratio and secondary characteristic fragment ions were used for qualitative analysis, and the excimer ion peak area was used for quantitative analysis. The results showed that all the drugs had good linearity in their respective concentration range, and the correlation coefficients was greater than 0.995. The average recoveries ranged from 70.3% to 116.2% at the addition levels of 10, 50 and 100 μg/kg. The relative standard deviation was 0.87% to 8.97%. The limit of detection was 1～10 μg/kg and the limit of quantification was 3～30 μg/kg. By constructing a first-order accurate mass database and a second-order spectral library, combine with retention time, accurate molecular mass number, isotope abundance ratio and secondary characteristic fragment ions, the method could achieve rapid screening and confirmation of target compounds. It had the advantages of simply, rapidity, high sensitivity, high selectivity and good precision, and was suitable for rapid screening and quantitative determination of multiple hormones in animal tissues.