In this study, we established a rapid method for intraoperative identification of thyroid carcinoma metastatic lymph nodes. We developed a rapid method of thyroid globulin (Tg) detection called Tg-POCT based on time-resolved fluorescence immunoassay and validated reagent performance. Thyroid and lymph node tissues with metastatic thyroid cancer were considered as the positive group, and muscle, fiber, fat, and other tissues were considered as the negative control group to verify the above method combined with fine needle puncture. CV of the same batch number was 8.38% and that of different batch numbers was 11.24%. The minimum detection limit was 0.02 ng/mL. The test conformity of tissue samples was 100% by using the self-designed reference value. Thus, Tg-POCT is feasible as a rapid intraoperative method to identify thyroid cancer metastatic lymph nodes.