High pressure homogenization (HPH) changes the activity and stability of some enzymes, improving characteristics of commercial enzymes. Glucose oxidase is a commercial enzyme applied to remove oxygen and glucose from food. This study assessed the HPH multiple passes on activity of glucose oxidase (GO). A volume of 3L of GO solution at pH 5.0 (3g.L-1) was homogenized at 100 and 150 MPa. Then, the samples were collected after 1, 2 and 3 consecutive passes on the equipment. The enzyme activities at 50 and 75ºC were measured, using a native GO as control sample. ANOVA and Tukey test were performed to determine the difference of samples results (p<0.05). Results at 50 ºC showed that HPH up to 100 MPa slightly alter the GO activity, but a significant activity loss (around 20%) was observed after three passes at 150 MPa (p<0.05). On the contrary the enzyme activity at 75ºC increased continuously after each passes at 100 and 150 MPa. The final residual activity reached (after 3 passes) was 60% (100 MPa) and 150% (150 MPa) higher than the observed for native sample (p<0.05). Thus, the results highlighted that multiple passes on homogenizer continuously changes the GO activity, which possibly is linked to alterations on GO structure caused by HPH. These data contributed to scientific knowledge about HPH effects on enzymes. Also, the results stand HPH process as a key step to be implemented on commercial glucose oxidase production, aiming to improve the enzyme activity especially at non-optimum temperatures.