Toxoplasmosis is a worldwide zoonotic disease caused by Toxoplasma gondii with harmful effects on animal and human health. Cross-sectional study was designed and conducted from January 2021 to March 2021 to estimate the prevalence and associated risk factors of toxoplasmosis among sheep and pregnant women in Debre Birhan city. Serum was collected from jugular vein of each sheep. Serum was also collected from pregnant women and exit interview was conducted with eligible mothers to obtain socio-demographic and behavioral data using structured questionnaires. All factors attributing to T. gondii infection in sheep and pregnant women were analyzed using logistic regression analysis. The seroprevalence of T. gondii infection in sheep was 46.3%. A significant association was observed between seroprevalence and sex, age and water source and significantly higher in female sheep (OR: 1.78, 95% CI: 1.16-2.74, P = 0.008), adult age (OR: 2.26, 95% CI: 1.46-3.49, P = 0.00) and river water (OR: 2.14, 95% CI: 1.22-3.74, P = 0.008). In pregnant women, the T. gondii seroprevalences were 61.2%. Multivariable logistic regression analysis showed that the risk of T. gondii infection was significantly higher in age of (35-36) (OR: 3.00, 95% CI: 1.57-5.75, P = 0.001), eating raw meat OR: 1.64, 95% CI: 1.03-2.59, P=0.036) spring water source (OR: 1.94, 95% CI:(3.29, P = 0.014) and contact with cats (OR: 1.68, 95% CI: 1.06-2.64, P = 0.027). This survey showed high seropositivity for T. gondii among sheep and pregnant women in Debre Berhan city. Therefore, further studies on isolation and genotype of the pathogen are crucial. It also requires action to prevent and control the infection in pregnant women, which poses a potential threat to a foetus. Key words: Pregnant women, Sero-prevalence, Sheep, T. gondii infection