Bile acids (BAs) are important regulatory factors of life activities, which are involved in the regulation of glucose, lipid and energy metabolisms, and closely associated with intestinal hormones, microbiotas and energy balance. BAs abnormalities easily lead to inflammation and metabolic diseases, in turn, the progress of diseases could influence characteristics of BAs. Therefore, accurate detection of BAs contents is of great significance to disease prevention, diagnosis and treatment. At present, the most widely used enzymatic method in clinical practice is applicable to the detection of total bile acid (TBA). In laboratory research, different types of BAs can be accurately separated and quantified by liquid chromatography-mass spectrometry (LC-MS). The metabolic profiling of BAs based on detection technologies can completely and accurately monitor their types and contents, playing a crucial role in disease prevention, diagnosis and treatment. We herein reviewed the main detection technologies of BAs and the application of metabolic profiling in related diseases in recent years.