Introduction: Tuberculosis (TB) is a major health problem, mainly in resource-limited settings. The aim of this study was to determine the prevalence of TB and rifampicin-resistant Mycobacterium tuberculosis(RR-MTB) among presumptive tuberculosis patients using Xpert MTB/RIF assay in Eastern Amhara, Ethiopia. Methods: A retrospective cross-sectional study was conducted among presumptive TB patients from three governmental hospitals in Amhara Regional State. Records of sputum sample results using Xpert MTB/RIF assay from January 2015 to December 2019 were investigated from registration books and analyzed using SPSS v.21. Results: Of the total of 26,656 (24,116 adults and 2540 children) TB presumptive patients included in the study, more than half, 14,624 (54.9%), were males and the median age was 36.87 (interquartile: 25.46–50.85 years). The majority of participants were new cases, 20,273 (76.1%), and with unknown HIV status, 18,981 (71.2%), respectively. MTB prevalence was 11% (95% CI: 9.34–12.08%) in all age groups, and 7.6% (95% CI 6.52–9.04%) among children. Of the MTB confirmed cases, prevalence of RR-MTB was 245 (8.3%) in adults and 14 (7.2%) in children. MTB infection was higher in the age groups of 18–35 years [adjusted odds ratio (AOR) = 2.17; 95% CI: 1.86–2.54, p< 0.001], 36–53 years (AOR = 1.31; 95% CI 1.11–1.54, p< 0.001), those who were relapse cases (AOR = 1.97; 95% CI 1.69–2.27, p < 0.0010), and failure cases (AOR = 4.67; 95% CI 3.36–6.50, p< 0.001). However, the age groups of 54–71 years (AOR = 0.79; 95% CI 0.65–0.95, p= 0.01) and over 71 years (AOR = 0.48; 95% CI 0.35–0.68, p < 0.001) were associated with lower MTB infection. Resistance to rifampicin was higher in the relapsed (AOR = 2.10; 95% CI 1.40–3.03, p< 0.001) and failure cases (AOR = 3.50; 95% CI 1.9–6.61, p< 001). Conclusion: Prevalence of MTB and RR-MTB low. TB infection was higher in adult age groups and those who had previous TB treatment history. Similarly, resistance to rifampicin was higher among the relapsed and failure patients. Appropriate measurements in monitoring of TB treatment could reduce TB and RR-MTB in the study area.