Abstract: OBJECTIVE To establish a method for the determination of theophylline residues in racehorse serum and urine by liquid-mass-isotope internal standard method. METHODS The strong cation exchange solid phase extraction was used to extract theophylline combined 6D-theophylline as internal standard. The Poroshell 120 SB-C₁₈(2.1 mm×50 mm, 2.7 μm) was chosen, with 0.1% formic acid(containing 5 mmol·L^–1 ammonium formate)-0.1% formic acid acetonitrile by gradient elution. The flow rate was 0.4 mL·min^–1. Electrospray source(ESI) ion source was used. Multi-reaction positive mode was used and ion pair of 181.1>124.1, 96.0 m/z and 187.1>126.9, 99.0 m/z were used for qualitative of theophylline and internal standard respectively. RESULTS Waters Oasis PRIME MCX 3CC/60 mg solid phase extraction column was optimal. The concentrations of serum and urine ranged 1.00–100.00 ng·mL^–1 and 2.50–100.00 ng·mL^–1 had a good linear relationship with the peak area(r² were 0.999 9 and 0.999 6, respectively). The detection limits were 0.30, 0.75 ng·mL^–1, and the quantitative limits were 1.00, 2.50 ng·mL^–1. The relative recoveries were 98.93%–114.49% and 94.85%–116.25%, respectively. The intra-batch precision RSD was≤3.81%, and the inter-batch precision RSD was ≤15.53%. The serum matrix had a strong inhibitory effect on theophylline, and the inhibition effect of urine was weak. CONCLUSION The method was proved to be simple, rapid, accurate and sensitive, and can be applied to the determination of theophylline residue in horse race doping control samples.