In this study, the serological surveillance of Rift Valley Fever virus (RVFV) in southern Egypt was carried out for 460 serum samples collected from domestic animals (unvaccinated), including cattle, sheep, goat, camel and donkey reared in three different provinces (Qena, Luxor and Aswan). Enzyme linked immunosorbent assay (ELISA) was used to detect RVFV antibodies. The results showed that 97 out of 460 animals were positive by using blocking ELISA. The percentage of RVFV infection in cattle, sheep, goat, camel and donkey was 5.55%, 65.21%, 14.44%, 20.65% and 0%, respectively. Geographical distribution and breeding system were taken into consideration for RVFV infection in these animals. The most prevalent type of infection was identified in intensive breeding farms systems (27.63%), and then in individual breeding systems (11.68%). Qena had a higher infection rate of RVFV (23.55%), in comparison to Aswan and Luxor (20.65% and 14.14%, respectively). Marked seroprevalence recorded in this study indicates a high incidence of infection in sheep (65.21%) and camel (20.65%); this necessitates the application of more effective strategies to control these types of infections in Egypt. This study provides a concise picture about the RVFV disease in southern Egypt. We need more similar studies targeted to clarify the reliable epidemiological status of RVFV disease in southern Egypt and other localities.