BACKGROUND: Thyroid nodules require pre-surgical cytological assessment for possible risk of malignancy. Many techniques were introduced to enhance differential diagnosis and to avoid unnecessary diagnostic surgery. OBJECTIVE: The study aims to investigate the potential use of ECM1 gene and MMP-2 protein as preoperative tumor markers in suspicious follicular thyroid lesions. METHODS: The study included 40 Egyptian cases with solitary thyroid nodules. They underwent preoperative FNAB followed by thyroidectomy. MMP-2 protein and ECM1 gene were detected using immunostaining and conventional semi-quantitative RT-PCR techniques; respectively. The diagnostic accuracy of FNAB, gene and protein expression level cutoffs was calculated by using ROC. RESULTS: Both MMP-2 protein and ECM1 gene expressions were significantly higher in malignant than benign group (P < 0.001). Both were significantly higher in higher tumor stages (PMMP-2= 0.002; PECM1 = 0.032) but only ECM1 significantly differed with tumor size (P < 0.006). The diagnostic performances of ECM1 expression scores was significantly better than that of FNAB (P = 0.049). A significant direct correlation was detected between ECM1 gene and MMP-2 protein expressions in cases of FVPC and of FC (P = 0.014). CONCLUSIONS: MMP-2 protein and ECM1 gene are useful preoperative markers for defining malignancy in suspicious thyroid nodules.