Zika virus (ZIKV) emerged as a global public health concern due to its relationship with severe neurological disorders. Non-structural (NS) proteins of ZIKV are essential for viral replication, regulatory function, and subversion of host responses. NS2B is a membrane protein responsible for the regulation of viral protease activity. This protein has transmembrane domains critical for the localization of viral protease to the endoplasmic reticulum membrane and a hydrophilic domain essential for folding, recruitment, and protease activity. Therefore, NS2B is considered a cofactor of viral protease which processes viral polyprotein and is essential for virus replication, making it an attractive antiviral drug target. Here, we report the backbone H, N, C resonance assignments of the full-length NS2B by high-resolution NMR. The backbone assignment will be necessary for determining the three-dimensional structure and backbone dynamics of NS2B, interaction mapping and screening potential of antiviral drugs against ZIKV and related pathogenic flaviviruses.