To explore new methods for intraoperative identification of parathyroid glands, 86 thyroid cancer patients, admitted to Xijing hospital from July 2017 to July 2018, were included. During lymph node dissection, parathyroid glands were firstly judged by clinician eyeballing, based on his clinical experience. Then, cytological detection was used for rapid identification via Diff-quik staining. PTH monitoring was performed by PTH detection kit. Finally, frozen pathology was examined and regarded as the golden standard. In this study, 172 suspicious parathyroid glands were observed. According to frozen pathology outcome, the accuracy, sensitivity and specificity of clinician eyeballing were calculated as 63.3%, 100%, and 13.9%. Kappa test showed poor consistency (kappa = 0.156), AUC area was 0.569 ± 0.045, 95%CI = (0.480–0.658), p = 0.123. For cytological and PTH detection, the accuracy, sensitivity and specificity were 91.7% vs. 92.3%, 93.6% vs. 93.8% and 89.0% vs. 90.3%. Kappa value was 0.829 vs. 0.842, indicating good consistency. AUC area was 0.908 ± 0.027 vs. 0.918 ± 0.025, 95%CI = (0.856–0.960) vs. (0.869–0.966), p < 0.001, indicating higher diagnositic value. Besides, compared with frozen pathology, cytological detection was easily and rapid. The time-taking between frozen pathology and cytological detection or PTH detection were 39.0 ± 6.59 min vs. 5.02 ± 0.78 min and 39.0 ± 6.59 min vs. 6.1 ± 1.23 min, p < 0.001. In conclusion, intra-operative cytological detection maybe potential for in-situ preservation of parathyroid glands.