N-nitrosamines (nitrosamines) are attracting increased attention because of their high toxicity and wide distribution. They have been strictly restricted by regulations in many fields. Researchers around the world have conducted substantial work on nitrosamine detection. This paper reviews the progress of research on nitrosamine detection methods with emphasis on biological-matrix samples. After introducing the category, toxicity, regulatory limit and source of nitrosamines, the paper discusses the most commonly used sample-preparation techniques and instrumental-detection techniques for nitrosamine detection, including some typical application cases.