The purpose of this study is to systematically investigate the pharmacokinetic (PK) behaviors of radix Sophorae tonkinensis (S. tonkinensis) using oxymatrine (OMT) and matrine (MT) as the target markers (2 mg/kg OMT and 1.3 mg/kg MT, oral administration). The PK characteristics in radix S. tonkinensis extracts were also compared with those of pure OMT. A fast ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) method was developed. OMT absorption was very fast, and no significant differences were observed (p > 0.05) in tmax, CL, and t1/2 for both pure OMT and extracts. Cmax and AUC0 → ∞ of pure OMT were significantly higher than those of S. tonkinensis extracts (Cmax, 61.64 ± 6.65 vs. 43.24 ± 10.14 ng/mL; AUC, 9894.48 ± 2234.99 vs. 4730.30 ± 3503.8 min ng/mL) (p < 0.05). However, the absolute OMT bioavailability of pure OMT was higher than that of the compound in radix S. tonkinensis extracts (6.79 ± 2.52% vs. 1.87 ± 2.66%). By contrast, the bioavailability of total alkaloids (OMT + MT) after pure OMT administration was 81.14 ± 8.83%, similar to that of radix S. tonkinensis extracts (69.36 ± 17.37%) (p > 0.05). It was presumed that OMT absorption has no effect on the bioavailability of the two alkaloids. Other constituents in radix S. tonkinensis extracts can influence the transformation of OMT to MT, which directly leads to variations in the PK behavior of OMT. In addition, the protein binding of OMT and MT in plasma was very low (4.80%–8.95% for OMT, 5.10–10.55% for MT). In conclusion, OMT in radix S. tonkinensis extracts exhibits different PK behaviors with pure OMT through the transformation of OMT to MT due to other complex ingredients.