West Nile virus (WNV) is a mosquito-transmitted single-stranded (ss) RNA flavivirus which causes West Nile infection and human disease of variable severity. Most of the people infected with WNV either have only minor signs or symptoms such as mild headache and fever or do not develop any signs or symptoms. Sometimes WNV causes a life-threatening illness as well as inflammation of the spinal cord or brain in infected patients. The genome ofWNV is single-stranded RNA (ssRNA) that contains the characteristics of positive polarity (mRNA sense). The strains of WNV are classified into two groups, such as lineage (1 and 2). The basis of grouping these strains based on the substitutions and deletions of signature amino acid in the envelope protein sequence. Among them, Lineage 1 WNV strains are responsible for developing the West Nile infection in humans. The genome of WNV consists of a single open reading frame (ORF) and it produced ten mature viral proteins which are classified into structural and nonstructural. Nonstructural protein 2B (NS2B) and Serine Protease (NS3) are two of nonstructural proteins of them. NS2B-NS3 has a major role in proteolytic processing of these nonstructural proteins. NS3 has several enzymatic activities like NTPase and RNA helicase. Among them,the helicase activities is one of them. There is evidence that Helicase C-terminal domain ofWest Nile Virus has a major role in the unwinding of dsRNA. But Helicase C-terminal domain is not yet explored, so our purpose is to investigate the physicochemical, structural, and functional features of Helicase C-terminal domain. Molecular modeling of the unexplored Helicase C-terminal domain was generated by using Phyre2 and SwissModel. The prediction of active ligand binding sites is generated by using PredictProtein server. This results that HelicaseC-terminal domain protein is stable and its nature is acidic which thermostability is high and has better hydrophilic property. By the putative transferase and catalytic functional activity, 14 binding sites were predicted. In Homology Modeling, it is revealed that 14 binding sites are predicted as ligand binding sites. This investigation decoded the role of this unexplored Helicase C-terminal domain of West Nile virus (WNV), and so it can pave the way for enriching our knowledge for pathogenesis and medication of WNV infection.