Background: Rhizoma Gastrodiae, the dried rhizome of Gastrodia elata Blume (G. elata) , is famous Chinese herb that belongs to the genus Gastrodia , family Orchidaceae. Gastrodin (GAS) is an effective monomer with one of the major active constituents in Rhizoma Gastrodiae . GAS has a good sedative and sleeping effect and has a mitigating effect on neurasthenia, insomnia, and headache. Moreover, in the circulatory system, it has the effect of reducing peripheral vascular resistance, blood pressure, and so on. Objectives: The objective is to investigate the pharmacokinetic characteristics after the administration of different doses of GAS by using pharmacokinetic method and make a preliminary judgment on whether the intake of GAS in the liver is involved in transporters. Materials and Methods: All healthy mice were randomly divided into three groups according to the drug concentration: low-concentration group (LC group), middle-concentration group, and high-concentration group. Then, each group received different doses of GAS by tail vein injection. The blood samples of different groups were harvested at different time points, and the blood drug concentration was evaluated by high-performance liquid chromatography method. The method was confirmed in terms of the linearity, precision, and accuracy. Results: The results showed that the analytical curve was linear over the concentration range of 4–40 μg/mL. In the intra- and inter-assay, the coefficient of variation was <7.23%. Moreover, the regression equation of the line was “Y = 49.43 X +0.027.” The results suggested that the elimination half-life period and area under the curve were slightly decreased, then non-linearly increased accompanying with increase of the dose of GAS. Conclusion: The results suggested that the pharmacokinetics of different dosage GAS administration were in accordance with the two compartment model in mice.