Autologous tooth transplantation is a treatment modality in which a donor tooth is transplanted into an extracted or surgically prepared alveolar socket of the same individual, thereby restoring a missing tooth. However, due to its high technical sensitivity, the clinical application of this procedure remains limited. Surgeons often encounter challenges in determining the optimal positioning and occlusion of the donor during surgery, which can result in excessive preparation of the alveolar bone and necessitate numerous crown adjustments. The aim of this study is to introduce a novel design 3D printed template that can assist in accurately placing donor teeth and simplify bonding procedures. It proposes that by utilizing 3D printed donor models combined with a 3D printed template, it is possible to reduce extra-oral time during autologous tooth transplantation, enhance preoperative evaluation and treatment accuracy, ensure optimal fit between donor and recipient alveolar sockets more effectively, minimize root absorption of transplanted teeth, promote post-transplantation healing processes as well as improve success rates and survival rates. In this paper, two cases involving autologous tooth transplantation guided by newly designed auxiliary positioning guides and adhesive guides are reported. The missing first or second molars were replaced with fully developed third molars with satisfactory one-year follow-up outcomes.