West Nile virus (WNV) is an enveloped RNA virus in the family Flaviviridae and belongs to Japanese encephalitis virus serocomplex group. The WNV has a wide geographic distribution that includes Africa, Europe, Asia, America and Australia. Recently, it has re-emerged as an important pathogenic organism, illustrated by the series of WNV outbreaks in North America and in Europe. Several hundred people are sacrificed by WNV infection every year. WNV can infect many mammals, birds, reptiles and amphibians. A variety of diagnoses for WNV infection have been developed, such as virus isolation, nucleotide amplification, antigen detection and serology. Flaviviruses, including WNV, share common nucleotide sequences and antigenic epitopes. Understanding these properties that can influence cross-reactivity is important for accurate diagnosis, especially because areas with multiple flaviviruses are currently expanding. Herein, the authors outline the different diagnostic methods for detecting WNV infection as well as important considerations in using these methods.