Abstract Background Hepatitis B serology is complex and a lack of knowledge in interpretation contributes to the inadequate levels of screening and referral for highly effective hepatitis antiviral treatments. This knowledge gap needs to be addressed so that current and future healthcare professionals are more confident in the detection and assessment of hepatitis B to improve the uptake of treatment and reduce long-term complications from the disease. Cartoons have been used effectively as a teaching tool in other settings and were considered as a potentially useful teaching aid in explaining hepatitis B serology. This study examines the impact of cartoons in improving healthcare professionals’ knowledge. Methods A cartoon based learning tool designed to simplify the complexities of hepatitis B serology was developed as part of an online learning program for medical practitioners, nurses and students in these professions. A retrospective analysis was carried out of pre and post online test results. Results An average improvement of 96% of correct answers to case study questions in hepatitis B serology was found across all ten questions following the use of an online cartoon based learning tool. Conclusion The data indicates a significant improvement of participants’ knowledge of hepatitis B serology from pre-test to post-test immediately following an online cartoon based learning tool. However, further research is required to measure its long term impact.