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In order to evaluate the pharmacokinetics of recombinant bovine interferon-alpha (rBoIFN-α) in cattle, which has potential for its antiviral and immunomodulatory activities, 12 animals of 6-month age were classified into 4 groups (n=3) to receive rBoIFN-α through IV, IM or SC routes at a dose of 5.0×103 IU/kg. Serum rBoIFN-α titer was evaluated using cytopathic effect (CPE) inhibition bioassay. Then, the standard pharmacokinetic parameters were calculated using the DAS (Drug and statistics) software. The concentration-time profiles of serum rBoIFN-α following IM administration, SC administration and IV administration were characteristics of the 1-, 1-, and 2-compartment open models, respectively. After a single dose of IV administration, the drug rapidly dispersed and was rapidly eliminated from the body (T1/2α=0.15±0.02 h, T1/2=6.48±0.49 h). After IM and SC administrations, the drug is rapidly absorbed and slowly eliminated from the body (For IM administration, Tmax=6.12±0.32 h, T1/2=8.19±0.74 h) (For SC administration, Tmax=4.06±0.56 h, T1/2=7.29±0.55 h). The bioavailability of rBoIFN-α after IM administration is 53.74%, which is higher than the bioavailability of SC administration (27.96%). Therefore, the results showed that the drug administration effect can be preferably obtained following a single dose IM injection using the rBoIFN-α aqueous preparation. We hope that this study will provide valuable information for the clinical application of rBoIFN-α as an potential antiviral agent.