During last two decades, more attention has been paid on finding new sources of fat and oil- as fuel or a part of human diet- because of population increase and then consumption. Oils and fats have been already produced from plant and animal sources, and according to their chemical composition they are considered as fuel or oil for human consumption. In recent years, biotechnologists have introduced some microorganisms as a new source for oil that it was based on their ability in production during short time with significant quantity. The current study was designed to produce oil from 2 cheap substrates- whey and hydrolysate mixture of wheat bran and straw- and their 1:1, 1:2 and 2:1 ratio by Aspergillus niger PTCC 5010. Based on mould nutritional requirements, some salts were added to media and pH adjusted to 6. After sterilization of all tested media, mould spores were washed from petri dish contains PDA and was inoculated on the mentioned media. Oil production curve, growth curve, nitrogen and sugar consumption curves were then drawn using 3 replicates of data. Regarding to drowen curves, the maximum oil production was in the fourth day after inoculation. During the first two days, nitrogen of media was almost depleted, but carbon source of all media tested decreased to minimum level in the fifth and sixth days. Biomass was increased with an ascendant rate up to fourth day, but after that was decreased rapidly and almost stopped because of nutrients limitation. Relation between substrate consumption with mould reaction in different times in each stage was evident by comparing of the curves. Results indicated that maximum content of oil was produced by mould in whey that was 23.28 percentage of dry biomass. This value for the media of hydrolysate and ratio of 1:1, 1:2 and 2:1 of whey and hydrolysate were 20.17, 21.89, 20.96 and 22.82, respectively. GC analysis demonstrated that produced oil in these media is contained palmitic acid (C16), stearic acid (C18), oleic acid (C18:1), linoleic acid (C18:2) and linolenic acid (C18:3). According to fatty acid profile obtained, it could be suggested that the produced oil by Aspergillus niger can be suitable as biodiesel. Maximum amount of oil production was in whey and maximum amount of unsaturated fatty acids was in hydrolysated of wheat bran and straw. Key words: microbial oil, biodiesel, Aspergillus niger , whey, hydrolysate of wheat bran and straw