Thiamine (vitamin B1) is essential to the health of all living organisms and deficiency has long been associated with diseases in animals such as fish, birds, alligators, and domesticated ruminant mammals. Thiamine is also implicated in several human diseases including Alzheimer's, diabetes, dementia, depression and, most notably, Wernicke-Korsakoff syndrome and Beriberi disease. Yet, highly sensitive and specific detection of thiamine remains an analytical challenge, as pM to nm levels of thiamine need to be detected in environmental and human samples, respectively, various phosphorylated variants need to be discriminated, and rapid on-site detection would be highly desirable. Furthermore, appropriate sample preparation is mandatory, owing to the complexity of the relevant sample matrices including fish tissues, ocean water, and body fluids. This Review has two objectives. First, it provides a thorough overview of analytical techniques published for thiamine detection over the last 15 years. Second, it describes the principles of analytical approaches that are based on biorecognition and may open up new avenues for rapid and high-throughput thiamine analysis. Most notably, periplasmic binding proteins, ribozymes, and aptamers are of particular interest, as they function as bioaffinity recognition elements that can fill an important assay technology gap, owing to the unavailability of thiamine-specific commercial antibodies. Finally, the authors provide brief evaluations of key outcomes of the major assay concepts and suggest how innovative techniques could help develop sensitive and specific thiamine analytical test systems.