DO3A-Thiol is widely used in the development of targeted molecular imaging agents, particularly in magnetic resonance imaging (MRI) and nuclear medicine. Its primary application lies in its ability to chelate metal ions such as gadolinium (Gd³⁺) or radioactive metals, forming stable complexes that can be conjugated to biomolecules like peptides, antibodies, or nanoparticles via the thiol group. This enables site-specific delivery of contrast agents to tumors or diseased tissues, enhancing imaging sensitivity and accuracy.

The thiol functionality allows for controlled bioconjugation under mild conditions, making it ideal for labeling sensitive biomolecules without compromising their biological activity. In theranostic applications, DO3A-Thiol-based constructs are used to combine diagnostic imaging and therapy in a single platform, especially in cancer research. Additionally, it supports the development of responsive or activatable MRI probes, where signal enhancement occurs only in the presence of specific biological triggers, improving early disease detection.