A cross sectional sero- and molecular survellience was conducted in Afar and Tigrayrnregions of Ethiopia to determine the seroprevalence, isolation and molecular identificationrnof Brucella species using Real time PCR in small ruminants and camels from Novemberrn2019 to May 2020. A total of 2523 serum samples; camel (n= 1625) and small ruminantsrn(n=898) were collected for the seroprevalence study. In addition, 15 vaginal swab samplesrnfrom animals having recent history of abortion were also collected for isolation andrnmolecular detection of Brucella species. Following serum harvest, the blood clot wasrnsubjected to DNA extraction for detection of Brucella species using PCR. In addition, datarnrelated to risk factors of brucellosis infection were collected to assess the potentialrnassociation of risk factors with seropositivity. Multi-species ELISA Kit was used to detectrnthe presence of circulating antibodies against Brucella species whereas; vaginal swabsrnwere processed and cultured on Brucella selective media for bacterial isolation andrnidentification. The seroprevalence of brucellosis was determined as 12.1% (95% CI: 10.1-rn14.5%) and 11.5% (95% CI: 8.9-11.8) for small ruminants and camels, respectively.rnMultivariate regression analysis revealed that sex, age and parity have significantlyrnassociated (p < 0.05) with Brucella infection in camels (OR 0.42 (95% CI: 0.27-0.64)rnP=0.000, OR 0.64(95% CI: 0.44-0.92), P= 0.017and OR 0.61(95% CI: 0.43-0.86), P=rn0.004 respectively. Where as, species, abortion history, and herd size were not statisticallyrnsignificant (P >0.05). Concerning small ruminants, sex, age, parity, abortion history, andrnherd size were not statistically significant (P >0.05). Bacterial culture result showed thatrnBrucella colonies were isolated from 13.3 % (2/15) of vaginal swab samples of caprine andrnboth isolates were identified as Brucella melitensis using real time PCR. Similarly 295rnblood clots from seropositive animals were tested with PCR and the result showed thatrnseven camel samples were positive for IS711 prmer that indicates Brucella genus. Furtherrnanalysis indicated that four of them were B. melitensis whereas three were not amplifiedrnwith both B. melitensis and B. abortus specific primers, which indicates they may be otherrnBrucella species. This study showed relatively higher prevalence of brucellosis in camelrnand small ruminants in Tigray and Afar Regions, signaling an urgent need for interventionrnto control the disease and prevent zoonotic transmission of brucellosis to the community.