Lactic acid is a natural organic acid and has many applications in the pharmaceutical, food, and chemical industries. It is used as acidulant and preservative, and recently its potential as substrate for the production of biodegradable plastic has been actively pursued. Lactic acid has been produced by fermentation of sugar-containing substrates including cheese whey using Lactobacillus helveticus, and Lactobacillus casei in most of the previous studies. Immobilization of cells was applied in most of these studies to improve the lactic acid yield. Bifidobacterium longum was used to produce lactic acid from cheese whey in this presentation. High lactic acid yield (0.82g/g) was obtained without immobilization of the cells. The fermentation conditions such as nutrient, pH, and temperature were optimized to obtain maximum lactic acid productivity. The results were also compared to that of other bacteria such as Lactobacillus helveticus.