Alginate as biopolymer consisting of β-D Mannuronic acid and α-L Guluronic acid monomer residues linked by (1-4) glycosidic bond. Brown seaweeds mostly Laminaria sp, Ascophyllum nodosum, Eclonia maxima, Sargassum sp. and Macrocystis pyrifera were earlier used for commercial alginate production. Major drawback of using sea weeds for alginate production were high production cost, impurity issue and safety concerns includes the reasons behind discovery of novel bacterial sources to make an efficient alginate production system. Later two bacterial genera Pseudomonas and Azotobacter were reported for alginate production. However, due to pathogenicity, and quality issue associated with some Pseudomonas strains, Azotobacter sp. was found to be most reliable and prioritized bacterial sources for alginate production with appreciable qualities which will be ready to be used for industrial applications. Therefore, current research aims to isolate and characterize a new strain of Azotobacter sp. with alginate-producing ability. Two different soil samples were collected from two places Deokali and Parsurampur, Jaunpur district in the spring (06 August 2022) and winter (06 February, 2023) seasons from Oryza sativa and Triticum aestivum rhizospheric region respectively. Azotobacter sp. was isolated by using Nitrogen Free Burk’s medium and alginate production was confirmed by gel formation assay method in the presence of CuSO4 and CaCl2 solutions. The carbohydrate qualitative test was done by using Molisch’s Test. Two novel strains Azotobacter chroococcum PU-RSD01 and Azotobacter chroococcum PU-RSD02 were isolated on the basis of high alginate producing ability. Morphological, biochemical and molecular techniques further characterized the isolates.