This study aimed to develop a new two-dimensional (2-D) ultrasound thyroid volume estimation equation using three-dimensional (3-D) ultrasound as the standard of reference, and to compare the thyroid volume estimation accuracy of the new equation with three previously reported equations. 2-D and 3-D ultrasound examinations of the thyroid gland were performed in 150 subjects with normal serum thyrotropin (TSH, thyroid-stimulating hormone) and free thyroxine (fT4) levels (63 men and 87 women, age range: 17 to 71 y). In each subject, the volume of both thyroid lobes was measured by 3-D ultrasound. On 2-D ultrasound, the craniocaudal (CC), lateromedial (LM) and anteroposterior (AP) dimensions of the thyroid lobes were measured. The equation was derived by correlating the volume of the thyroid lobes measured with 3-D ultrasound and the product of the three dimensions measured with 2-D ultrasound using linear regression analysis, in 75 subjects without thyroid nodule. The accuracy of thyroid volume estimation of the new equation and the three previously reported equations was evaluated and compared in another 75 subjects (without thyroid nodule, n = 30; with thyroid nodule, n = 45). It is suggested that volume of thyroid lobe may be estimated as: volume of thyroid lobe = 0.38.(CC.LM.AP) + 1.76. Result showed that the new equation (16.9% to 36.1%) had a significantly smaller thyroid volume estimation error than the previously reported equations (20.8% to 54.9%) (p < 0.05). There was a significantly larger thyroid volume estimation error when thyroid glands with nodules were examined (p < 0.05). With the use of the appropriate thyroid volume equation, 2-D ultrasound can be a useful alternative in thyroid volume measurement when 3-D ultrasound is not available.