Objective To explore the application of personalized guide plate combined with intraoperative real-time navigation in repairing of mandibular defect using fibula muscle flap, providing the basis for the precise repair and reconstruction of mandible. Methods The clinical data of 12 patients (9 males and 3 females) aged from 23 to 71 years (mean, 55.5 years) between July 2019 and December 2021 were recorded. These patients were diagnosed as benign or malignant mandibular tumors, including 2 cases of ameloblastoma, 6 cases of squamous cell carcinoma, 2 cases of osteosarcoma, 1 case of adenoid cystic carcinoma, and 1 case of squamous carcinoma. All patients were treated with mandibular amputation, and then repaired by double-stacked three-segment fibula muscle flap. Preoperative virtual design scheme and guide plate were performed. During the operation, personalized guide plate combined with real-time navigation was used for fibular osteotomy and shaping. Thin-slice CT examination was performed at 2-3 weeks after operation, and was fitted with the preoperative virtual design scheme. The difference between the distance of bilateral mandibular angles relative to the reference plane in three-dimensional directions (left-right, vertical, and anterior-posterior) and the difference of the medial angle of the lower edge of the mandible reconstructed by fibula were measured, and the mean error of chromatographic fitting degree was calculated. Results The guide plate and navigation were applied well, and the fibula shaping and positioning were accurate. The fibula muscle flap survived, the incision healed well, and the occlusal relationship was good. All 12 patients were followed up 1-29 months, with an average of 17 months. There was no significant difference on the distance of bilateral mandibular angles relative to the reference plane in the left-right [(−0.24±1.35) mm; t=−0.618, P=0.549], vertical [−0.85 (−1.35, 1.40) mm; Z=−0.079, P=0.937], and anterior-posterior [(−0.46±0.78) mm; t=−2.036, P=0.067] directions. The difference of the medial angle of the lower edge of the mandible reconstructed by fibula was also not significant [(−1.35±4.34)°; t=−1.081, P=0.303)]. Postoperative CT and preoperative virtual design fitting verified that there was no significant difference in the change of the mandibular angle on both sides, and the average error was (0.47±1.39) mm. ConclusionThe personalized guide combined with intraoperative real-time navigation improves the accuracy of peroneal muscle flap reconstruction of the mandible, reduces the complications, and provides a preliminary basis for the application of visual intraoperative navigation in fibula muscle flap reconstruction of the mandible.