Background: The long-term effect of three-dimensional printed heart model (3DPHM) on knowledge acquisition of congenital heart disease (CHD) remains unknown. This prospective cohort study aims to investigate the role of 3DPHM in improving immediate knowledge gain and long-term knowledge retention on CHD among the medical students. Methods: Fifty-three second and third year medical students were assigned into two groups to compare their immediate knowledge acquisition and knowledge retention after an education session on anatomy and pathophysiology of CHD. During the 1.5 hour-long education session, both the control (n = 25) and study groups (n = 28) had access to identical teaching materials: digital 3D heart models, 2D diagrams, and medical images, except for 3DPHM which were only used in the study group. The immediate knowledge gain was assessed via an online quiz, whereas the long-term knowledge retention was assessed using another quiz in 6-weeks’ time post-intervention. A survey was also conducted to evaluate the participants’ learning experience. Results: There is no significant difference in the immediate knowledge acquisition and long-term knowledge retention between the groups (U = 272, p = 0.16 and r = –0.143, p = 0.15 respectively). Majority of the students (96% in control group and 85% in 3DPHM group) responded that the 3DPHM would have/had improved their learning experience. Despite that, there is no significant difference in the self-perceived knowledge improvement between the groups. Conclusions: This study concludes that the 3DPHM do not significantly improve both immediate knowledge acquisition and knowledge retention among the medical students. However, further research with larger sample size, as well as categorizing the type of questions in the quiz, is needed to better assess the role of 3DPHM in different educational components.