Acetolactate synthase (ALS) or Acetohydroxyacid synthase (AHAS) catalyzes the first step in the synthesis of the branched-chain amino acids i.e., valine (2-amino-3-methylbutanoic acid), leucine (2-amino-4methylpentanoic acid), and isoleucine (2-amino-3-methylpentanoic acid), in plants, bacteria, algae and fungi but not in humans. AHAS is the main target enzyme for sulfonylurea active ingredients; Amidosulfuron, Nicosulfuron and Cyclosulfuron those assist in lowering branched-chain amino acid synthesis through inhibition to form the complex of Lactyl-ThDP(TDL) to ALS with great practical importance. Amino acid composition, evolutionary and sequence analysis of the ALS protein from Arabidopsis thaliana and its homologous were systematically studied. Composition analysis reveals that ALS is a soluble protein. Moreover, the phylogenetic tree showed different clusters based on the source organism and multiple sequence alignment depicts conservative nature in amino acid residues. Furthermore, molecular docking has been conducted to study the interactions between ALS of Arabidopsis thaliana and TDL in presence/absence of the active ingredients of sulfonylurea herbicide groups. Molecular docking studies confirm active ingredients are effective to inhibit the binding of TDL to ALS. Our obtained results can be very useful to study specific protein interactions along with developing new herbicides using computational methods.