Flavivirus are single-stranded, enveloped positive-strand RNA viruses. Increased emerging viral infections are attributed to factors including sero-cross-relativity, migration, climate change, urbanization, commercial exchange activities and tourism, and nature of the geographical areas. These factors have led to the spilling over of these pathogens to susceptible urban settings and previously unexposed geographic regions. Therefore, some of the individuals experience co-infection, which is a simultaneous infection of a human being by more than one virus species at the same time. Co-infection in a patient makes the scenario difficult by interfering in isolation of the targeted pathogen from the sample for diagnosis. Co-infection with JE has been linked with dengue and Zika virus infection. Co-infections are associated with several of the challenges including differential diagnostics. Upon co-infection, one virus may interfere with the replication of the other virus co-infected and may even alter its virulence, which ultimately affects the outcome of the disease. Therefore, this chapter focuses on the co-infection of Flavivirus and its associated challenges.