Brucellosis is one of the serious infectious bacterial diseases endemic in Egypt, as it makes great economic losses because it makes high rates of abortion and infertility. This work intended to detect the seroprevalence of animal Brucellosis the New Valley governorate. From March 2022 to May 2023, a cross-sectional examination investigating the seroprevalence of Brucellosis in various animals was carried out in the five centers of the New Valley governorate, Egypt. 954 serum samples from various animals (250 cattle, 100 camels, 335 sheep, and 269 goats) were collected in total. According to a serological analysis of serum samples, the prevalence of Brucellosis in cattle, camels, sheep, and goats using the Rose Bengal Plate test (RBPT) was, respectively, (8, 12, 14,6, and 16.7%). Summer was the season with the highest Brucellosis prevalence (42.6%). El-Dakhla has the highest level of Brucellosis prevalence (26.3%). In sheep, goats, cattle, and camels, the prevalence of Brucellosis was highest in the age groups of (<2, ≤1, ≤3, and <3-5 year), respectively. Moreover, the prevalence of Brucellosis was greater in females than in males (13.5 and 11.7%, respectively). As a result, age, locality, sex, and season are the most important danger elements that can significantly affect the seroprevalence of animal Brucellosis.