The paper aims to explore the studying method for the pharmacokinetics of drugs in target organs, the pharmacokinetic process of tramadol hydrochloride in the extracellular fluid of frontal cortex (FrCx) of mice was investigated.  Six male mice (Kunming strain) were anaesthetized (urethane, 1.8 g·kg⁻¹, ip) and secured on a stereotaxic frame.  A microdialysis probe was implanted into the FrCx and perfused with artificial cerebrospinal fluid at a flow rate of 2 μL·min⁻¹.  One hour later, mice were administrated (ip) with tramadol hydrochloride (50 mg·kg⁻¹) and dialysates were collected continuously at 12-min intervals (24 μL each) for 6 h.  The tramadol concentration in dialysates was determined by HPLC-Ultraviolet detection method, and the concentration-time curve and pharmacokinetic parameters of tramadol were calculated with DAS software.  The results showed that the pharmacokinetic process of tramadol in the FrCx extracellular fluid of mice was fitted to a two-compartment open model, and the main pharmacokinetic parameters t_1/2α , t_1/2β , t_max , C_max and AUC_0−∞ were (0.27 ± 0.05) h, (2.72 ± 0.24) h, (0.50 ± 0.10) h, (2 110.37 ± 291.22) µg·L⁻¹ and (4 474.51 ± 441.79) µg·L⁻¹·h, respectively.  In conclusion, a studying method for pharmacokinetics of drugs in the target organ is established, which is simple and feasible.  Tramadol hydrochloride shows a two-compartment model in the extracellular fluid of the mouse FrCx, and the distribution- and elimination half-life are 0.5 h and 2.7 h, respectively.