Gelsenicine is an indole alkaloid isolated from Gelsemium elegans Benth. In recent years, the role of G. elegans Benth preparations in anti-tumor, analgesic, dilatation and dermatological treatment has attracted attention, and it has been applied clinically, but it is easy to cause poisoning with its use. An UPLC-MS/MS method was established to determine the gelsenicine in mouse blood, and the pharmacokinetics of gelsenicine after intravenous (0.1 mg/kg) and intragastric (0.5 and 1 mg/kg) administration was studied. Deltalin was used as internal standard; a UPLC BEH C column was used for chromatographic separation. The mobile phase consisted of acetonitrile and 10 mmol/L ammonium acetate (0.1% formic acid) with a gradient elution flow rate of 0.4 mL/min. Multiple reaction monitoring mode was used for quantitative analysis of gelsenicine in electrospray ionization positive interface. Proteins from mouse blood were removed by acetonitrile precipitation. A validation of this method was performed in accordance with the US Food and Drug Administration guidelines. In the concentration range of 0.05-100 ng/mL, the gelsenicine in the mouse blood was linear (r > 0.995), and the lower limit of quantification was 0.05 ng/mL. In the mouse blood, the intra-day precision RSD was <12%, the inter-day precision RSD was <15%, the accuracy ranged from 89.8 to 112.3%, the average recovery was >76.8%, and the matrix effect was between 103.7 and 108.4%, which meet the pharmacokinetic research requirements of gelsenicine. The UPLC-MS/MS method is sensitive, rapid and selective, and has been successfully applied to the pharmacokinetic study of gelsenicine in mice. The absolute bioavailability of gelsenicine is 1.13%.