Rapid and sensitive detection of clinical biomolecules in a multiplexed fashion is of great importance for accurate diagnosis of diseases. Mass spectrometric (MS) approaches are exceptionally suitable for clinical analysis due to its high throughput, high sensitivity, and reliable qualitative and quantitative capabilities. To break through the bottleneck of MS technique for detecting high-molecular-weight substances with low ionization efficiency, the concept of mass spectrometric biosensing has been put forward by adopting mass spectrometric chips to recognize the targets and mass spectrometry to detect the signals switched by the recognition. In this review, the principle of mass spectrometric sensing, the construction of different mass tags used for biosensing, and the typical combination mode of mass spectrometric imaging (MSI) technique are summarized. Future perspectives including the design of portable matching platforms, exploitation of novel mass tags, development of effective signal amplification strategies, and standardization of MSI methodologies are proposed to promote the advancements and practical applications of mass spectrometric biosensing.