This study was conducted to determine the seroprevalence of Coxiella burnetii in cows from Assiut Governorate, Egypt. A total of 268 blood samples were collected from cows (176 females and 92 males). The age of these animals ranged between 2 months and more than 3 years. Screened for C. burnetii using indirect enzyme-linked immunosorbent assay. A total of 15.67% (42/268) was serologically positive. The seropositivity was high in females (19.56%) than males (13.63%). The native breed was more infected (17.44%) than Fresian (13.33%) and mixed ones (11.76%). In cows, a higher seroprevalence was observed in the age group more than 1 to 2 years (22.38%). Dairut city recorded the highest seroprevalence rate (40.74%) of C. burnetii. C. burnetii seroprevalence was higher in hot season (20.10%) than cold season (5.95%). Cows free from ticks recorded higher seroprevalence (22.58%) than those infested with ticks (12%). Cows with fever and pneumonia recorded higher seroprevalence (14.29%) compared to animals with other signs. In conclusion, findings of this study revealed the wide spread of C. burnetii infection among cows at Assiut Governorate, Egypt.