Calcitonin (CT) measurement on fine-needle aspiration cytology (FNAC) washouts (FNAC-CT) is a promising tool in the diagnosis of medullary thyroid carcinoma (MTC). Guidelines for the method with codified cut-off are needed to use this technique in clinical routine. This study was undertaken to assess the best pre-analytical procedure and to define a reliable reference value for FNAC-CT. Fifty-four patients underwent thyroid surgery, so MTC was excluded by surgical pathology examination and CT immunostains. Before surgery, FNAC-CT was measured on 78 thyroid nodules from such 54 patients. Needles were rinsed by normal saline and specific CT-free dilution buffer, and CT was measured by a fully automated immunochemiluminometric assay. FNAC-CT levels were not significantly different in normal saline or specific buffer. The calculated 97.5th upper FNAC-CT value was 8.5 pg/mL (saline) and 7.43 pg/mL (buffer), respectively. Seeing its relatively large sample size, rigorous selection criteria and inclusion of CT immunostaining of thyroid nodules, the present study provides a reliable guideline and a clinically relevant reference value for FNAC-CT measurement in thyroid nodules.