Thyroxine (T4), as a type of thyroid hormone (TH), is a key hormone in regulating human metabolism, growth and development, central nervous system functions, and energy balance. It relies on TH transport proteins to reach cells and exert its biological actions. However, the binding of nitrophenol pollutants to TH transport proteins prevents the delivery of thyroid hormones to cells, thereby inhibiting the effects of the hormones. This study combines spectroscopic experiments and computational simulations to explore the mechanism of nitrophenols' interference with TH transport proteins. Detailed information on the quenching mechanism, binding parameters, interaction forces, binding models, and conformational changes of nitrophenols (PNP), chlorinated nitrophenols (CNP), and brominated nitrophenols (BNP) with TH transport proteins is obtained through spectroscopic experiments. Nitrophenols are found to form hydrogen bonds with residues Lys15, Arg378, and Arg381, respectively, thereby displacing T4 at the binding site in the TH transport proteins. With an increasing number of halogen atoms, the affinity of halogenated nitrophenols for TH transport proteins intensifies. Computational simulations are used to further understand the binding modes and binding sites, providing molecular-level insights into the binding of NPs in the cavity of TH transport proteins. Theoretical evidence from molecular docking and molecular dynamics (MD) simulations supports the experimental findings.