Background: We aimed to investigate the ability of carbon nanoparticles to identify parathyroid glands with vasculature during thyroid surgery. Material and methods: Totally 42 patients with various thyroid diseases were selected for the prospective research of carbon nanoparticle injection used in thyroidectomy. Another 42 patients without receiving carbon nanoparticle injection were selected as the control group. All cases underwent total or subtotal bilateral thyroidectomy. Before the ligation of the superior and inferior poles of the thyriod lobes, 0.1 mL of a carbon nanoparticle suspension was injected into the two poles. Important tissues such as the recurrent laryngeal nerve, parathyroid gland with blood supply and the inferior thyroid artery were identified and protected. Results: The parathyroid glands with vasculature were not stained and thus remained the primary color in all cases, while abnormal thyroid tissues were stained black. After 5 minutes, thyroid lobes were injected with the carbon nanoparticle suspension and the original color of parathyroid glands was unchanged. Even the parathyroids with blood supply can be identified and protected. The number of parathyroid glands for autotransplantation was one in the test group and six in the control group. At one day after the thyroid surgery, hypocalcemia became detectable in four patients of the test group and in five of the control group. Twenty-four instances of hypoparathyroidism occurred at 1 day after surgery in the control group, while ten instances in the test group. Conclusion: Intraoperatvie carbon nanoparticle suspension injection is an effective and safe technique for guiding thyroid surgery. The carbon nanoparticle suspension plays an important role in identifying the vasculatural parathyroid glands, while protecting the physiologic function of the parathyroid glands during surgery.