Used as a redox indicator in analytical chemistry, methylene blue trihydrate plays a key role in titrations where it changes color based on the oxidation state of the solution. It is widely applied in biological staining to differentiate cells and cellular structures under the microscope, especially in bacteriology and histology.

In medicine, it serves as an antidote for methemoglobinemia by helping to convert methemoglobin back to hemoglobin. It is also used in photodynamic therapy, where it acts as a photosensitizer to destroy pathogens in blood products or treat certain infections.

In aquaculture and veterinary medicine, it functions as an antifungal and antiparasitic agent for fish and eggs. Additionally, it finds use in dyeing textiles and paper due to its strong coloring properties, and in water treatment processes.