The objective of this study was to investigate ractopamine residues in urine, plasma and hair of cattle during and after treatment. Three cattle (body weight = 620 ± 6.2 kg) were administered ractopamine (2.01 mg/kg body weight) into the rumen for 5 consecutive days. Ractopamine concentrations in samples were determined by an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS-MS) method. The concentrations of parent ractopamine in urine were 1,631.1 ng/mL on withdrawal day 0 and 8.3 ng/mL on withdrawal day 14. After hydrolysis of its conjugates, ractopamine concentration ranged from 11,796.7 ng/mL (withdrawal day 0) to 39.7 ng/mL (withdrawal day 14). In plasma, parent ractopamine and its conjugates were below the limits of quantification (LOQ = 0.2 ng/mL) on withdrawal days 5 and 7. Accumulation of ractopamine in black and white hair was 124.6 and 78.1 ng/g, respectively, on withdrawal day 0, and 226.7 and 165.6 ng/g, respectively, on withdrawal day 14. This study demonstrated the rapid elimination and high bioavailability of ractopamine in urine and plasma in cattle. However, accumulation of ractopamine in cattle's hair is high and persistent, so hair can be used as the target matrix for monitoring ractopamine abuse in ruminants.