Abstract: Sodium gluconate was widely used in food and other industries.The effect of oxygen supplement, which was regulated through areation and agitation, on fermentation time and yield of gluconic acid was investigated.The results showed that compared to the control, only increasing agitation could enhance oxygen supplement significantly, shorten fermentation time from 35.7h to 21.4h and improve yield from 72.88% to 87.24% with 300g /L initial glucose concentration and 0.8vvm areation. In contrast, only increasing aeration had minor effect on fermentation time and yield when the agitation was 300r /min, just shorten to 30.5h and improved to 80.76%, respectively.As a consequence, combination of increase of agitation and aeration simultaneously to markedly enhance oxygen supplement, the best performance was achieved with 20.8h for fermentation time and 88.47% for yield.