To evaluate the bioavailability of puerarin sustained release tablet (SR-Tab.) and Yufengningxin tablet (YU-Tab.), a liquid chromatography method was developed and validated to determine puerarin in dog plasma. Chromatographic separation was performed on Diamonsil C18 column using a mixture of methanol–acetic acid–water (25:6:69, v/v/v) delivered at a flow rate of 1.0 ml/min and detected by UV. 4-Hydroxybenzaldehyde was used as the internal standard. The linear range for puerarin was from 60 to 1800 ng/ml (r = 0.9991) with a limit of quantitation of 60 ng/ml. Within-day accuracy and precision ranged from −3.0 to 2.2% and from 1.2 to 4.3%, between-day accuracy and precision ranged from −4.1 to 2.6% and from 1.3 to 5.7%, respectively. The mean extraction recoveries of puerarin determined over the three concentrations were (90.3 ± 5.2)%, (95.7 ± 1.4)% and (93.1 ± 3.5)%. A significant difference was observed in main pharmacokinetic parameters of Tmax, Cmax and AUC0–∞ between puerarin SR-Tab. and YU-Tab. in dogs. The smoother plasma concentrations were obtained from SR-Tab. in dogs and the results were as expected.