Abstract: One-factor-at-a-time and orthogonal array design methods were employed to investigate the effect of bovine blood sample pre-treatment methods on the detection results of clenbuterol residues by Enzyme-linked Immunosorbent Assay (ELISA) .The results indicated that the serum sample quality was effectively improved, ELISA assay for detection bovine blood clenbuterol residual false positive results were reduced at the greatest degree, by controlling standing time of blood sample before centrifugation and after centrifugation, proportion of added anticoagulants into bovine blood.The effect of bovine blood sample pre-treatment methods on detection of clenbuterol residues were in the following order:standing time of blood sample before centrifugation>proportion of added anticoagulants into bovine blood>standing time of blood sample after centrifugation.The optimum bovine blood sample pre-treatment methods were for standing 30min before centrifugation, added anticoagulants in proportion of 1:9, standing 0min after centrifugation.In accordance with the bovine blood sample optimal pre-treatment conditions before analysis, two positive samples were screened out of 30 bovine blood samples using ELISA, which was consistent with the results from LC-MS/MS analysis.Within the linear range of 1.0 ~ 8.1μg/L, the false positive rate of clenbuterol residues was 0%.