Background: Most of the smallholder animal farmers in rural Bangladesh depend on livestock for their livelihoods but significant percentage of these animals do not achieve their potential mainly due to inadequate nutrition and disease and occasionally transmit zoonotic disease like brucellosis. Brucellosis has been recognized as a neglected zoonotic disease in the low-income countries that produce few or no clinical signs in the affected animals making it more difficult for the dairy farmers to use preventive measures. However, sero-monitoring could help to detect the occurrence of Brucella infection in smallholder dairy farm management system. Objective: This study aimed to determine the prevalence and associated risk factors for positivity of bovine brucellosis by using sero-screening and milk ring test supported with questionnaire Materials and Methods: A cross-sectional study on bovine brucellosis was conducted in smallholder dairy cows in the district of Mymensingh during the period from August to December 2019. Serum samples of 460 lactating cows along with their milk samples were collected randomly. Serum samples were screened for brucellosis with Rapid Antigen Kit Test and Rose Bengal Test (RBT), whereas milk samples were tested with Milk Ring Test (MRT). Farm and animal level demographic and risk factor data were collected using a questionnaire and analyzed using univariable and multivariable logistic regression. Results: The overall sero-prevalence was found to be 3.9% (95% CI 2.4-6.2) using RBT and Rapid Antigen Kit Test and 2.8% (CI 1.5-4.9) using Milk Ring Test, respectively. The odds of brucellosis was 7.4 times (95% CI: 2.5-21.5) higher in cows with repeat breeding that without repeat breeding. Moreover, the sero-prevalence of brucellosis was significantly higher (Odds ratio: 15.7; 95% CI: 5.2-47.4) in cows with retention of fetal membranes than without retention of fetal membranes. Conclusions: The prevalence of Brucella infection in smallholder dairy farms with no adaptation of any preventive measures against this disease in Bangladesh. The sero-prevalence of brucellosis recorded in this study should be interpreted with caution and confirmatory diagnosis is needed to know the accurate status of brucellosis in smallholder dairy farms. The prevalence of Brucella infection in smallholder farms by using sero-test and milk ring test warrants further molecular test prior to embarking on a control program.