The gluconic acid batch fermentation was conducted using mutant Aspergillus niger NCIM 530 strain under submerged condition in 50 L semiautomatic stirred-tank fermenter. Certain cost-effective source as golden syrup was effectively utilized instead of glucose for successful industrial fermentation process. The significant level of gluconic acid (85.2 gL-1) production was observed with maximum 86.97% glucose conversion over 44 hours. This process provides great advantages over traditional submerged fermentation strategies and substrates, as showed by effective production of gluconic acid by utilizing novel substrate as a golden syrup. To reduce analysis time with better accuracy, an effort has been made to use a method for evaluation of parameters like conversion of substrate and production of gluconic acid during the fermentation by using High Performance Thin Layer Chromatography (HPTLC).