Leaf spot disease caused by the fungus Alternaria brassicae is a major threat to mustard production worldwide. This disease affects all parts of the mustard plant, including leaves, stems, and pods, leading to reduced yield and quality of the crop. Conventional methods of identifying leaf spot disease in mustard by Alternaria brassicae have their advantages and limitations. Preceding methods of detecting leaf spot disease in mustard caused by Alternaria brassicae have several limitations including visual inspections, time-consuming processes and need for trained personnel. To overcome these issues, Molecular techniques, remote sensing, image processing, and biosensors have all proven to be effective in accurately detecting and quantifying leaf spot disease in mustard. In order to gain a better understanding of this pathogen, the present study aimed to investigate the morphological, cultural, growth characteristics, and molecular aspects of the Alternaria brassicae isolate I-1B20. As the result of this research, the radial growth and sporulation of the leaf spot of mustard is present in large numbers. In culture medium of OMA, its growth is high with best sporulation results. The pH and temperature in the plant is 8.0 and 24°C of best sporulation and radial growth results. The humidity rate of the growth of the plant is 100. This information will aid in the development of effective management strategies for controlling leaf spot disease in mustard plants. The sequences, BLAST, genetic expression and phylogenetic tree are performed through the isolate of the Alternaria brassicae to develop the disease resistant strategies.