Age of seed culture for fructooligosaccharides (FOS) production using Penicillium sp. H12 at optimal conditions for shaked flask culture was found non significant for FOS yield. But in fermentor, the seed age of 18th hour yield the highest FOS. Addition of 50 g/l sucrose at the 9th and 12th hour gave the best yield of FOS. But addition of sucrose at 100, 150 and 200 g/l sucrose reduced FOS yield significantly. Sucrose addition continuously at 1 ml/min could overcome the concentration effect. With continuous addition, even total addition of 400 g of sucrose still increased FOS yield as compared to the non added one. Enzyme response for FOS production was found mainly as an extracellular enzyme. The highest enzyme activity was detected at the 12th-15th hour of culture. FOS purification was done successfully by using activated carbon column eluted with 15% ethanol. The recover of more than 95% was achieved with at least 4 repeating usage.