Hyaluronic acid (HA) was depolymerized by a combination of ultrasound, hydrogen peroxide and copper ion. The structures of high-molecular-weight hyaluronic acid (HMW-HA) and low-molecular-weight hyaluronic acid (LMW-HA) were determined by Fourier transform infrared (FTIR) spectroscopy, circular dichroism (CD) spectroscopy, and UV-VIS absorption spectroscopy. The degradations of HMW-HA using a physical method, a chemical method, and a combination of physical and chemical method were compared. The results show that HA can be effectively degraded by a combinatorial method involving ultrasound, hydrogen peroxide, and copper ion. Under the degradation conditions of 50 mM H₂O₂, 5.0 μM CuCl₂, 160 W, pH 4.0, and reaction at 50 °C for 30 min, the content of glucuronic acid was 36.56%, and the yield of LMW-HA was 81.71%. The FTIR, CD, and UV-VIS absorption spectra of HA did not change with the decrease in molecular weight, indicating that the structure of HA remained intact during the degradation.