Glucose oxidase have get pronounced attention due to its wide range of uses in different industries including chemical, pharmaceutical, food, beverage, biotechnology and clinical analysis. Present investigation deals with N-nitroso-N-methylurea (NMU) chemical that was used as mutagen for enhanced glucose oxidase (GOX) production from the wild strain of Aspergillus niger. After mutagenesis and screening methods, TS-NMU-100 mutant derived isolate of Aspergillus niger screened out as best positive mutants with the 260.10% increase in glucose oxidase activity corresponding to its wild strain. Production of GOX from wild and NMU mutant derived strains of Aspergillus niger was carried out by using pre-optimized media conditions using corn steep liquor (CSL) as substrate. It was found that N-nitroso-N-methylurea, chemical mutagen could be efficiently used for the hyper-production of GOX from A. niger. It was also revealed that NMU mutant strains of Aspergillus niger (A. niger) having a strong capability of GOX production and could be valuable for further advance research investigations and commercial scale production of GOX especially in food and pharmaceutical preparations.