In recent years, the incidence of nodular goiter and thyroid carcinoma (TC), as well as the amount of surgery, have been increasing simultaneously. To summarize the significance and experience of combined nano-carbon tracer and nano-fluorescence assay for parathyroid misresection reduction in thyroid surgery. The data of patients who underwent thyroid surgery from November 2016 to July 2017 in the Thyroid Disease Diagnosis and Treatment Center of the Tenth People’s Hospital of Tongji University were collected and divided into test group (combined nano-carbon tracer and nano-fluorescence assay method, 80 cases) and a control group (nano carbon tracer method, 80 cases). Comparison of the patient data and preoperative and postoperative blood calcium, blood phosphorus, PTH value in the control and test group was performed. No significant difference was found on gender, age, preoperative Calcium, Phosphorus, Magnesium and PTH, postoperative Calcium, Phosphorus and Magnesium was found in the control and test group (all the p > 0.05), whereas significant difference was found on the postoperative PTH between control and test group (p < 0.001). Moreover, significant reduced number of parathyroid misresection was found in the test group compared to the control group (p = 0.003). Significant difference could be found on pre- and postoperation data of blood Calcium, Phosphorus, Magnesium and PTH in both control and test group (all the p < 0.05). A combined nano-carbon tracer and nano-fluorescence assay can be used in thyroid surgery, which is helpful to distinguish parathyroid tissue, avoid accidental injury or missection of parathyroid gland as much as possible, and reduce the incidence of postoperative hypocalcemia.