Methylene blue is a fluorescent dye discovered in 1876 and has since been used in different scientific fields. Only recently has methylene blue been used for intraoperative fluorescent imaging. Here, the authors review the emerging role of methylene blue, not only as a dye used in clinical practice, but also as a fluorophore in a surgical setting. We discuss the promising potential of methylene blue together with the challenges and limitations among specific surgical techniques. A literature review of PubMed and Medline was conducted based on the historical, current and future usage of methylene blue within the field of medicine. We reviewed not only the current usage of methylene blue, but we also tried to grasp its' function as a fluorophore in five main domains. These domains include the near-infrared imaging visualization of ureters, parathyroid gland identification, pancreatic tumors imaging, detection of breast cancer tumor margins, as well as breast cancer sentinel node biopsy. Methylene blue is used in countless clinical procedures with a relatively low risk for patients. Usage of its fluorescent properties is still at an early stage and more pre-clinical, as well as clinical research, must be performed to fully understand its potentials and limitations.