Abstract: Protein biomarkers exist in human cells and peripheral circulatory system. Changes in their concentrations often reflect the organism in a state of health or disease. The low abundance of protein biomarkers in biological organisms, coupled with the fact that some proteins exist in bound forms, increases the difficulty of detection. Highly sensitive and accurate quantification of protein biomarkers is a common goal in clinical medicine and life sciences research. Traditional detection methods, such as chemiluminescence immunoassay and electrochemiluminescence, struggle to meet the requirements for multi-component analysis and accurate quantification of protein biomarkers due to issues like cross-reactivity and spectral overlap inherent in these techniques. In contrast, Immuno-mass spectrometry (IMS), which combines immune recognition and mass spectrometry detection, demonstrates high specificity, sensitivity, and multi-component analysis capabilities, can be used for the structural analysis, as well as the accurate quantification of multiple proteins simultaneously. IMS has become an important tool for the structural analysis and quantitative analysis of target molecules in complex matrices and is widely used in the detection of protein biomarkers. This article reviews the characteristics and research progress of three different types of IMS, analyzes the critical issues for the sensitivity, accuracy, and stability of IMS detection, introduces the application of IMS in protein biomarker detection, and discusses the challenges and further development of IMS.