Open AccessObjective: The quality blood grouping reagents is clearly an important factor for blood transfusion and diagnostics and many international standard Anti-B reagents are available for blood grouping in India and there is a need of indigenously developed, cost effective potent Anti-B secreting monoclonal antibody which can be used as a standard blood grouping reagent. To develop indigenous, cost effective standard Anti-B reagent which can be used in the scale up system to ensures constant supply which will ultimately be cost-effective if produced in-house. Method: Murine anti-B monoclonal antibodies have been produced by hybridoma fusion with specificity for human blood group B following the immunization of BALB/c mice with group B cells. Results: Three hybridoma clones were developed with specificity to human B blood group. Out of three hybridoma clones two of them, show potent titer, avidity, and sensitivity to be used as blood typing reagent. One clone, 3D5D7G2 reacted well in screening tests with an avidity of < 4 seconds with B cell. Conclusion: This IgM antibody reacted with 100% accuracy when tested with more than 2500 samples of A, B, O blood groups and satisfies all the laid down criteria under WHO guidelines to be labeled as an Anti-B reagent. The suitability of these antibodies as blood grouping reagents is discussed.