Purpose <p>This study aims to investigate a precise and effective preoperative localization and marking technique for parathyroid mass to enhance surgical accuracy and efficiency, minimize exploration time, and achieve consistency between preoperative localization and intraoperative findings.</p> Methods <p>This study enrolled 69 patients scheduled for parathyroidectomy. All participants underwent preoperative laboratory tests (blood calcium and serum parathyroid hormone [PTH] levels etc.) as well as high-frequency ultrasound. Each patient also underwent at least one functional imaging modality, including Technetium-99&#xa0;m sestamibi (⁹⁹ᵐTc-MIBI) scintigraphy and/or Positron Emission Tomography–Computed Tomography (PET-CT). Following informed consent, nanocarbon was injected under ultrasound guidance into the superficial capsule of the target parathyroid tissue for preoperative staining.</p> Results <p>The study cohort comprised 69 patients, including 15 males and 54 females, with a mean age of 53.4 ± 13.1 years. Preoperative ultrasound demonstrated benign parathyroid lesions in all patients. Functional imaging was performed as follows: ⁹⁹ᵐTc-MIBI alone (<i>n</i> = 56), PET-CT alone (<i>n</i> = 46), or both (<i>n</i> = 36). Under ultrasound guidance, a total of 115 target lesions were preoperatively injected with nanocarbon. Intraoperative observations revealed that 111 lesions were successfully stained. Staining failed in the remaining four: two showed no staining, and two exhibited staining only in the adjacent thyroid tissue. This yielded an overall staining success rate of 96.5% (111/115). The median maximum lesion diameter was 16.85&#xa0;mm (IQR: 11.20&#xa0;mm). Histopathological analysis confirmed 55 parathyroid adenomas, 58 hyperplastic parathyroid glands, one lymph node, and one adipose tissue focus. The nanocarbon localization strategy combined preoperative imaging and intraoperative nanocarbon staining. The positive predictive value (PPV) of using successful nanocarbon staining concordant with preoperative imaging as the criterion for identification was 98.3% (113/115). No serious complications were observed following nanocarbon injection. All patients exhibited normalized PTH levels (&lt; 88 pg/mL) on the first day after surgery.</p> Conclusion <p>The ultrasound-guided nanocarbon injection technique, by providing clear visual guidance, serves as a valuable adjunct in parathyroid surgery.</p>