According to the requirement of the calibration in minimally invasive blood glucose monitoring, a method based on microdialysis was presented to monitor glucose level in interstitial fluid continuously. An experimental system simulating the continuous change of glucose concentration in vivo was built. The influences on recovery of microdialysis caused by flow rate, glucose concentration, and temperature etc. were studied. The results led to the conclusion that the recovery fell by 71.7% when perfusion rate increased from 0.3 μL/min to 3.0 μL/min, while the different concentrations of glucose solutions scarcely contribute to the recovery instead, and the temperatures from 25 to 58 °C caused the recovery to increase by 34.6%.