Despite the high success rate of endodontic microsurgery (EMS), it is difficult to suggest EMS as a general treatment option considering the difficulty of the procedure. A surgical guide has been proposed to overcome this problem. This study aimed to evaluate the stability of the surgical guide of a new design concept, as well as the accuracy of root resection, and to introduce the manufacturing method of the newly designed surgical guide. The experiment was conducted on 59 roots (9 in the maxillary and 50 in the mandibular region) of adult human cadavers. The surgical guide was designed using CAD/CAM design software based on cone-beam computed tomography (CBCT) images and optical scan files. Unlike conventional surgical guides, the surgical guide proposed herein was designed to act as a tooth-bone-supported removable appliance. Two different types of guides were prepared: the osteotomy guide (O guide) for separation of the cortical bone above the root tip with a trephine bur with an outer diameter of 6 mm and the root resection guide (R guide) for resection of the root tip with a trephine bur with an outer diameter of 4 mm. For stability evaluation, the guides were pressed at five predetermined locations after installation and checked for the presence of any movement. For accuracy evaluation, the length at which the root tip was cut was measured and examined by overlapping the preoperative and postoperative CBCT images. Of the 15 R guides, 14 were stably installed without mobility. For the R guide group, the root tip was resected with an average of 3.2 mm, showing better results than the no-guide group with an average of 4.0 mm. The newly designed surgical guide of this study can be applied more stably, enabling root resection to be performed more accurately and simply according to the preoperative plan than when performed without a guide.