This study assessed the possibility of using iodine-containing fertilizers for agronomic biofortification of rye biomass used as fodder for cows, and establish the best application method and form and the optimal dose of iodine (I) under field conditions. The impact of iodine fertilization on grain iodine content was not studied. Results showed that agronomic biofortification of rye plants with iodine, influenced by its dose, form, and method of application was highly effective in increasing I shoot contents. Plant I-enrichment via foliar and soil application significantly affected I concentration in plant biomass even at a low dose (2.5 kg ha−1). Soil I application as KI appeared optimal for rye plants used as fodder for cows, especially cropped under the soil with a neutral reaction. Iodine application improved the biological quality of rye plants by increasing concentrations of sugar, chlorophylls, and at a low rate, protein and total antioxidant capacity.