Citric acid is in greater demand globally. Aspergillus niger is a workhorse for the production of citric acid. Cane molasses have been chosen as a novel and economically viable substrate because of the recently rising demand for citric acid and the necessity for substitute materials as substrates. Therefore, this study was conducted to improve citric acid production from A. niger using by-product of sugar (sugarcane molasses) through gamma radiation. A. niger was isolated from soil and the fermentation process was performed in shaking flasks using sugarcane molasses. Data revealed that significantly highest production of citric acid (31.30 ±0.17 g/L) was obtained in a medium containing (15%) substrate level. the maximum citric acid productivity was achieved at pH 6. Spore suspensions of A. niger were subjected to different doses of gamma radiation. Results clarified that the maximum amount (42.82 ±0.62 g/L) of citric acid was observed at 1.0 KGy. The amount of acid produced was increased by 1.11 fold higher than control by the addition of 3% (v/v) ethanol after 24 hour of fermentation time. Moreover, the addition of 0.15% K4Fe (CN)6 enhanced citric acid manufacturing. Furthermore, citric acid productivity from immobilized gamma irradiated A. niger spores was increased by 1.2 times more than that of the free ones. Citric acid was separated from the fermentation broth by precipitation method using CaCl2. In addition, FT-IR Analysis of the extracted citric acid from A. niger confirmed that there is no change in the main characteristic absorption bands of the standard citric acid.