Objective To establish an HPLC approach for determining the plasma drug concentration of evodiamine (EVO) in evodiamine novel nano emulsion (ENNE) in rats, and to investigate the pharmacokinetics and bioequivalence of ENNE in rats. Methods Twelve SD rats were evenly randomized into two groups and were administered intragastrically with ENNE (containing EVO 100 mg/kg) or EVO (100 mg/kg). The plasma drug concentrations of EVO were measured at 5 min, 10 min, 15 min, 30 min, 45 min, 1 h, 2 h,5 h, 8 h, 12 h, 24 h, 48 h and 72 h after administration of ENNE or EVO by HPLC. The chromatographic conditions were as following:the mobile phase was methanol and 0.1% of formic acid-water solution (66:34,V/V), the flow rate was 1 mL/min, the injection volume was 100 μL, and the detection wavelength was 225 nm. The concentration-time curve was drawn by excel software, and the main pharmacokinetic parameters and bioequivalence were calculated by DAS 2.1.1 software. Results The established method was fast, accurate, and had good linear correlation. The AUC_0-∞ of ENNE and EVO were (8 248.88±69.92) μg·h·L^-1 and (884.82±83.52) μg·h·L^-1, and the t_1/2 of ENNE and EVO were (1.70±0.60) h and (1.05±0.45) h, respectively. The AUC_0-∞ of ENNE was 9 times that of EVO, and the t_1/2 of ENNE was 1.62 times that of EVO.ENNE and EVO were not bioequivalent. Conclusion Bioavailability and absorption of ENNE are higher than EVO, and ENNE and EVO are not bioequivalent.