Abstract: The response surface methodology was employed to optimize the process conditions of Gluconobacter frateurii for producing glycolic acid. First, single factor test and Plackett-Burman test design were used to screen out the three main factors affecting the production of glycolic acid: Sorbitol concentration, yeast powder concentration, and ethylene glycol concentration. On this basis, the steepest climbing path method was used to approach the maximum response value range, and the response surface methodology was used to determine the interaction and optimal conditions between these main factors. The results showed that the optimization concentration were: Sorbitol 40.30 g/L, yeast powder 36.90 g/L, CaCO₃ 2.50 g/L, ethylene glycol 28.14 g/L, culture temperature 30 °C, pH7, inoculum size 10% (v/v), rotating speed 200 r/min, fermentation period 48 h. Under the optimized condition, the titer of glycolic acid reached 21.04 g/L. After three parallel experiments, the actual average titer was close to the predicted titer. The conversion rate of glycolic acid was increased by 28.25%, reaching 74.77%, and the productivity was 10.52 g/(L·d).