Objective: To investigate the clinical application value of the new tracer mitoxantrone hydrochloride in imaging of cervical lymph nodes and identifying of parathyroid, recurrent laryngeal nerve during thyroid cancer radical surgery. Methods: A prospective controlled study was conducted between January 2022 and April 2024 at Tianjin First Central Hospital, recruiting 180 patients with thyroid cancer, including 54 males and 126 females, aged from 26 to 69 years. The patients were randomly divided into three groups: the mitoxantrone hydrochloride lymph node tracing group (MHI group), the nanocarbon lymph node tracing group (nanocarbon group), and the control group without lymph node tracers, with 60 cases in each group. All patients underwent total thyroid resection and regional lymph node dissection. The number of detected lymph nodes and positive metastatic lymph nodes and surgical field clarity scores and the identification rate of parathyroid glands and the instances of inadvertent excision were compared between the groups. Serum calcium and parathyroid hormone (PTH) levels at different perioperative time points and treatment-related complications were analyzed. One-way ANOVA, chi-square test and two independent sample non-parametric tests, were employed for statistical analyses. Results: The mean operation time, neck drainage volume and hospital stay in the MHI group and the nanocarbon group were respectively significantly lower than those in the control group (P<0.05). The mean staining intensity score, tracer success rate and surgical field clarity score in MHI group were respectively significantly higher than those in nanocarbon group (P<0.05). The mean numbers of dissected lymph nodes positive metastatic nodes and identification rates of parathyroid glands in MHI group were respectively significantly higher than those in both the nanocarbon and control groups (P<0.05). The rates of inadvertent parathyroid excision in both MHI group and nanocarbon group were significantly lower than those in control group (P<0.05). On postoperative day 1 and day 5, mean serum calcium and PTH levels in MHI group were respectively significantly higher than those in nanocarbon group and control groups. However, on postoperative day 1, there was no difference in mean serum calcium levels between the nanocarbon group and the control group (P>0.05), though PTH level in the nanocarbon group was higher than that in the control group. By postoperative day 5, both serum calcium and PTH levels were higher in the nanocarbon group compared to the control group (P<0.05). On postoperative days 30, there were no differences in serum calcium and PTH levels between the MHI group and the nanocarbon group (P>0.05). The risks of facial numbness, hand and foot tetany and choking during drinking were lower in both the MHI and nanocarbon groups compared to the control group (P<0.05). Conclusion: MHI demonstrates advantages in rapid targeted delivery and clear staining of the surgical field during regional lymph node dissection in radical thyroidectomy for thyroid cancer, effectively reducing the risk of collateral damage to the recurrent laryngeal nerve and parathyroid glands.