Measurement of gene expression levels in thyroid tumor cells in aspirates was difficult because it is interfered with peripheral blood cells or infiltrating lymphocytes. In this study, we established a novel method to separate thyroid tumor cells from blood cells efficiently with mesh filtration. The expression level of trefoil factor 3 (TFF3) mRNA was estimated using LGALS3 mRNA as an internal control (T/G ratio) in 148 preoperative thyroid aspirates. Intra-assay coefficients of variation (CV) of T/G ratio for high, moderate, and low samples were 6.5%, 2.5%, and 9.7%, respectively, and inter-assay CV for high, moderate, and low samples were 27.7%, 21.9%, and 38.2%, respectively. Nondiagnostic samples in terms of T/G ratio and cytology were 12.2% and 16.9%, respectively. We observed no interference with the data by contaminating blood cells. Among these patients, 12 patients received more than two repeated aspirations. We did not observe a marked day-to-day variation except in two cases. All 13 preoperative aspirates diagnosed as malignant by cytology showed an extremely low T/G ratio, whereas 93 aspirates diagnosed as benign by cytology showed extremely varied T/G ratios and 21.5% of them showed a T/G ratio below the cut-off value. Eleven cases underwent surgery. All nodules showing a low T/G ratio were diagnosed as papillary carcinoma by pathological diagnosis. However, one nodule diagnosed as follicular adenoma after surgery showed a high T/G ratio. Our present method may be a promising preoperative test for measuring mRNAs in thyroid aspirates.