The pathological progression of thyroid diseases poses a serious threat to human health. Because thyroid diseases are closely related to selenocysteine (Sec), it is necessary to investigate the relationship between Sec and thyroid diseases. Herein, we design and synthesize a ratiometric near-infrared fluorescent probe (Mito-Cy-Sec) to analyze the fluctuations and roles of Sec in cells and in mice thyroid diseases model. The probe is composed of a near-infrared heptamethine cyanine fluorophore, an acrylamide as the response moiety, and a lipophilic triphenylphosphonium cation as the mitochondrial localization group. After reacting with Sec for 5 min, the probe Mito-Cy-Sec exhibits a distinct ratiometric fluorescence signal accompanied by a color change from green to blue. The applicability of Mito-Cy-Sec in mitochondrial localization is assessed via the super-resolution imaging. Mito-Cy-Sec has been successfully applied to detect the fluctuations of Sec concentration in human thyroid epithelial/cancer cell lines (Nthy-ori-3 cells/BHT101 cells) and mice thyroid disease (thyroiditis and thyroid cancer) models. Besides, both of our probes Mito-Cy-Sec and commercial ROSGreen H2O2 are employed to examine the interrelationship between H2O2 and Sec in cells and in mice models. The results demonstrate that the relevant-levels between H2O2 and Sec are exactly negative correlation. The related-levels of Sec and H2O2 may be identified as diagnostic indicators for the auxiliary diagnosis of thyroid diseases. We suppose that our probe Mito-Cy-Sec can be employed as a promising chemical tool for the diagnosis of thyroid diseases.