Bioassay functions, which are provided by the International Commission on Radiological Protection, are used to estimate the intake activity of radionuclides; however, they include considerable uncertainties in terms of the internal dosimetry for a particular individual. During a practical internal dose assessment, the uncertainty in the bioassay function is generally not introduced because of the difficulty in quantification. Therefore, to clarify the existence of uncertainty in the bioassay function and provide dosimetrists with an insight into this uncertainty, this study attempted to quantify the uncertainty in the thyroid retention function used for radioiodine exposure. The uncertainty was quantified using a probabilistic estimation of the thyroid retention function through the propagation of the distribution of biokinetic parameters by the Monte Carlo simulation technique. The uncertainties in the thyroid retention function, expressed in terms of the scattering factor, were in the ranges of 1.55–1.60 and 1.40–1.50 for within 24 h and after 24 h, respectively. In addition, the thyroid retention function within 24 h was compared with actual measurement data to confirm the uncertainty due to the use of first-order kinetics in the biokinetic model calculation. Significantly higher thyroid uptakes (by a factor of 1.9) were observed in the actual measurements. This study indicates that consideration of the uncertainty in the thyroid retention function can avoid a significant over- and under-estimation of the internal dose, particularly when a high dose is predicted.