A new method using on-line cleanup technology combined with liquid chromatography tandem mass spectrometry( UHPLC-MS / MS) was developed for the determination of seven kinds of β2-agonists residues,Formoterol, Salmeterol, Carbuterol, Clenisopenterol, Clenpenterol, Clencyclohexerol and Clenbuterolhydroxymethyl in livestock manure. The sample was sufficiently extracted by acidic acetonitrile and diluted by0.2% formic acid. The extract was online purified on Hyper Sep Retain CX column where the sample matrix was washed away and the analytes were retained. The analytes were eluted into Hypersil Gold C18 column by2% Ammonia-methanol solution. The seven β2-agonists were detected in selected reaction monitoring( SRM)mode via positive electrospray ionization( ESI+) by liquid chromatography-tandem mass spectrometry. The results showed good linearity correlation coefficients over 0. 9928 for the seven kinds of β2-agonists in 0. 5-100 μg / L concentration range. The LOD of seven kinds of β2-agonists in livestock mature is 1 μg / kg,while the LOQ is 5 μg / kg. When 5-50 μg / kg of the seven kinds of β2-agonists were added into the blank livestock manure,an average recovery of 67%- 112% was obtained with the relative standard deviations of 2. 9%-10.2%. The method is simple,rapid and has good reproducibility for quantitative and confirmatory analysis ofβ2-agonist residues.