Sensitive and accurate detection of biomolecules by multiplexed methods is important for disease diagnosis, drug research, and biochemical analysis. Mass spectrometry has the advantages of high sensitivity, high throughput, and high resolution, making it ideal for biomolecular sensing. As a result of the development of atmospheric pressure mass spectrometry, researchers have been able to use a variety of means to identify target biomolecules and recognize the converted signals by mass spectrometry. In this review, three main approaches and tools are summarized for mass spectrometry sensing and biopsy techniques, including array biosensing, probe/pen-based mass spectrometry, and other biosensor–mass spectrometry coupling techniques. Portability and practicality of relevant mass spectrometry sensing methods are reviewed, together with possible future directions to promote the advancement of mass spectrometry for target identification of biomolecules and rapid detection of real biological samples.