Background: The occurrence of high titres of alpha (anti-A) and beta (anti-B) haemolysin immunoglobulin G antibodies in blood causes haemolysis during blood transfusion from a group O donor, commonly and inappropriately known as the ‘universal blood donor’, to a group A, B or AB recipient. Surprisingly, haemolysin testing is not routinely done during blood transfusion services in Bamenda, Cameroon.Objective: This study aimed to determine the prevalence of haemolysin among blood group ‘O’ donors at the Regional Hospital Bamenda Blood Bank, Bamenda, Cameroon.Methods: This was a cross-sectional descriptive study carried out between June and September 2020 at the Regional Hospital Bamenda Blood Bank, Bamenda, Cameroon. Blood group O donors who were free from transfusion-transmissible infections were selected systematically and serially and their serum tested for the presence of haemolysin. Haemolysin titres were determined, and titres ≥ 8 were considered significant. The associations between haemolysin prevalence and age group, gender and Rhesus D blood group were determined using the chi-square test.Results: The prevalence of haemolysin among the 480 study participants was 52.1% and significant haemolysin titres were detected in 18.5%. There was no association between haemolysin and gender, age group or the Rhesus D blood group.Conclusion: The prevalence of significant titres of haemolysin among participants in this study was high. There is the need to test for haemolysin in blood group O donors to prevent the potential risk to blood group A, B, and AB recipients and to provide safer blood for transfusion.