Objectives This study was conducted to disclose the specific antibodies against M. bovis of bovine tuberculosis (bTB) in blood and milk serum samples, with detection of the most prevalent clinical signs in positive cows. Methods In some rural areas of two Iraqi provinces (Wasit and Dhi-Qar), 119 lactating cows were submitted to the clinical examination with obtaining of blood and milk to tested by using the IDEXX ELISA test. Results The overall seroprevalence in blood and milk was (20.16%) and (15.12%), respectively. In Wasit, the prevalence was (22.85%) and (15.71%), while in Dhi-Qar, the prevalence was (16.32%) and (14.28%) in blood and milk, respectively. As well as, marked significant differences in seroprevalence were observed between and within the two study's provinces and samples. According to clinical examination, a significant rising (P > 0.05) was revealed in respiratory disorders, decreasing in milk production, emaciation, rough hair coat and repetitive reproductive problems, whilst a significant decreasing (P < 0.05) in persistent feces abnormalities, mastitis, lymph nodes enlargement and loss of appetite. Conclusion The study demonstrated, for the first time in Iraq, the efficient of IDEXX ELISA, as a screening test in the detection of bTB in lactating cows by using blood and milk serum samples, and the competence of milk, as sample, in exhibition of infection. Also, the study exposed the high infection rate of bTB in cows of rural areas of Wasit and Dhi-Qar provinces.