Cervical mediastinoscopy is useful for diagnosing lung and mediastinal disease. Ultrasound is a safe real-time diagnostic tool widely employed in many surgical fields. Ultrasound was used in cervical mediastinoscopy in our cohort with satisfactory results. This study investigated the safety, feasibility, and availability of video-assisted mediastinoscopy (VAM) combined with ultrasound for mediastinal lymph node biopsy.A total of 87 cases involving cervical mediastinal lymph node biopsy performed from November 2015 to May 2020, with complete clinical and pathological information, were retrospectively analyzed in the Department of Thoracic Surgery at Beijing Chaoyang Hospital. The cohort was divided into two groups: ultrasound-guided biopsy under video-assisted mediastinoscopy (UVAM) (44 cases) and routine VAM (43 cases). Operation time, biopsy number and nodal stations, postoperative complications, pathological conditions, and surgical difficulty were compared between the two nodal stations.UVAM was significantly shorter and more lymph node specimens were obtained than with VAM. There was one case of fatal bleeding and two cases of right recurrent laryngeal nerve injury in the VAM group, and no postoperative complications in the UVAM group.When used with cervical VAM, ultrasound guidance assists physicians assess the space between lymph nodes, surrounding tissues, and large vessels systematically, making biopsy safer and easier, improving lymph node sampling, and decreasing postoperative complications. Furthermore, surgeons can easily learn and master this method.Significant findings of the study: Ultrasound was used in combination with cervical mediastinoscopy and the results showed that ultrasound guidance makes biopsy in patients safer and easier, improves lymph node sampling, and decreases postoperative complications.Surgeons can easily learn and master this method.