Milk contains key nutrients in optimal proportions, which makes it to be an important component of the human diet. Despite the fact that consumers receive with milk a significant amount of readily available nutrients, milk can be a source of dangerous factors of chemical and biological origin. The level of physiological hormones found in milk is not a limiting factor and is studied solely for scientific purposes. However, milk hormones can have a negative effect on the health of consumers. Based on this, the aim of this work was to study the concentration of triiodothyronine and thyroxine in colostrum, milk and blood plasma of cows at different stages of lactation. The secretion of the mammary gland and blood plasma of cows of Ukrainian black-and-white dairy breed, in their 2nd–5th lactations with milk yield 5100–5700 kg of milk per previous lactation was the object of the study. The concentration of triiodothyronine and thyroxine using enzyme-linked immunosorbent assay was investigated in withdrawn samples. The obtained data indicates that the highest level of secretion of iodine-containing thyroid hormones with milk is observed on 1st–4th days of lactation. Thereafter, the concentration of hormones in milk statistically significant decreases by 2.8–3-fold (P ˂ 0.001) till 30–40th day of lactation. The high concentration of iodine-containing hormones in colostrum allows to enhance metabolic processes in calves. Studies of the concentration of thyroid hormones in the blood plasma of cows showed a gradual increase from the beginning of lactation till 10th–14th and then till 30th–40th days of lactation. By 10th–14th days we observed 2.2-fold growth of triiodothyronine level (P ˂ 0.001), and till 30th–40th days this parameter has increased by another 12.5 %. The level of thyroxine on 10th–14th days of lactation was higher, compared to such in the beginning of lactation by 62.2 % (P ˂ 0.001), and by 30th–40th day increase was by 40.9 %. The main reasons for such changes are the formation of lactation dominant, the discharge of hormones secreted by the breast and the stimulation of metabolic processes during lactation. The prospects of further research lie in investigation of the dependence of triiodothyronine and thyroxine levels in the secretion of the mammary gland on productivity, breed, age and diet of animals.