Itaconic acid is an industrially crucial organic acid due to its broad range of applications. The main hurdle in itaconic acid production is the high cost of the substrate, i.e., pure glucose, required for the fermentation process. Pakistan annually produces about 1.7 to 1.8 million metric tonnes of mango fruit. Keeping this in view, the potential of a sugar-rich fruit by-product, i.e., mango peels, was analyzed to be used as a substrate for the biosynthesis of itaconic acid using Aspergillus niger by submerged fermentation. Different physicochemical parameters (incubation period, temperature, agitation rate, inoculum size, and pH) were optimized using the central composite design (CCD) design of response surface methodology (RSM). The maximum production of itaconic acid, i.e., 4.6 g/L, was analyzed using 10% mango peels w/v (water hydrolysate), 3 mL inoculum volume after 5 days of fermentation period at pH 3, and a temperature of 32 °C when the media was kept at a 200-rpm agitation speed. The itaconic acid extraction from mango peels was done using the solvent extraction method using n-butanol. The identification and quantification of itaconic acid produced in the study were done using the Fourier Transform Infrared Spectroscopy (FTIR) spectrum and the High-Performance Liquid Chromatography (HPLC) method. According to HPLC analysis, 98.74% purity of itaconic acid was obtained in the research. Hence, it is concluded from the results that sugar-rich mango peels can act as a promising substrate for the biosynthesis of itaconic acid. Further conditions can be optimized at the bioreactor level to meet industrial requirements.