This paper proposes an innovative microfluidic device for hyaluronic acid (HA) extraction and electrochemical detection utilizing PMMA based microchip that integrated with gold nanoelectrode ensemble (GNEE). Gold nanoelectrode ensemble is fabricated using electroless deposition in a thin polycarbonate (PC) film and then directly bonded onto the PMMA substrate for high performance electrochemical detection. In the study, microwave is firstly used to decrease the interference of protein on the working electrode, and then electrostatic force is used to separate and extract HA in the rat serum sample. The used of GNEE electrodes in the microchip not only enhances the signal but also decreases the background noises, resulting a high detection limit of 0.1 mg/mL for HA, and showing good linear responses in the concentration range of 10 mg/mL-0.1 mg/mL (R2=0.956). The proposed microchip device provides a fast detection method for hyaluronic acid analysis.