Background and Aims Methylene blue hydrochloride injection (MHI) for tracing is a new type of tracer approved by the National Medical Products Administration for use in thyroid cancer. There are few related reports at present, leaving significant research gap. Therefore, this study compared the effects of MHI and carbon nanoparticles as tracers on lymph node dissection and parathyroid protection in robot-assisted thyroid cancer surgery, so as to provide new tracer options for clinical work.Methods Using a prospective, randomized controlled design, patients undergoing da Vinci robot-assisted total thyroidectomy in the Department of Thyroid and Breast Surgery, the 960th Hospital of the People's Liberation Army were enrolled between June 2022 to June 2023. Grouping was done using a random number table. Patients receiving MHI were included in the study group, and patients receiving carbon nanoparticles were included in the control group. The number and tracing rate of level Ⅵ lymph nodes in the two groups were recorded and compared. The total number and positive number of level Ⅵ (± lateral neck) lymph node dissections, preoperative and postoperative 1-d and 6-month levels of blood calcium and parathyroid hormone (PTH), rate of inadvertent parathyroidectomy, incidence of hypoparathyroidism, incidence of temporary hypocalcemia after surgery, and whether there were residual marks at the injection site on the skin after surgery were analyzed to compare the differences in the effects of the two tracers.Results A total of 99 patients were included in this study, with 7 lost to follow-up. Finally, 92 patients completed the trial, with 46 in each group. There were no significant differences in general conditions between the two groups (all P>0.05). The lymph node tracing rates in the Ⅵ region were 97.1% (95% CI=95.7-98.5) in the study group and 96.4% (95% CI=94.9-97.8) in the control group, with no significant difference between the groups (P>0.05). The difference in the lymph node tracing rate in the Ⅵ region between the two groups was 0.7% (95% CI=1.2%-2.8%), and the Ⅵ region lymph node tracing rate in the study group was non-inferior to that in the control group (95% CI lower limit ≥-10%). The total number of lymph nodes detected in the Ⅵ region was 557 and 630 in the study and control groups, respectively (Z=-0.388, P=0.698), and the number of positive lymph nodes detected was 125 and 92, respectively (Z=-1.443, P=0.149). There were no statistically significant differences in preoperative and postoperative 1-d and 6-month PTH and blood calcium levels between the two groups (all P>0.05). Inadvertent parathyroidectomy occurred in one case in the study group and 4 cases in the control group, with no significant difference between the groups (P>0.05). There was no significant difference in the incidence of transient hypoparathyroidism after surgery between the two groups (P>0.05), but the incidence of transient hypocalcemia in the study group was lower than that in the control group (P<0.05). There was no permanent hypoparathyroidism in the study group, while one case occurred in the control group. One patient in each group had residual marks on the skin after surgery. During the 6-month follow-up, the marks had disappeared in the study group patient but remained in the control group patient. There were no cases of recurrence during the follow-up period.Conclusion In da Vinci robot-assisted thyroid cancer surgery, the use of MHI for tracing can achieve lymphatic tracing and parathyroid protection effects similar to those of carbon nanoparticles suspension injection.