Abstract: Ultrasensitive analysis of biomolecules provides a necessary tool for the accurate understanding of the internal mechanism of biological activities in all living things. According to its high sensitivity, high throughput, strong qualitative ability and rapid analysis speed, MS plays an important role in the detection of biomacromolecules and a variety of metabolites. Because of the complex matrices and limited sensitivity, it is still challenging to precisely analyze trace biological samples or even single cells by mass spectrometry. Therefore, combined with appropriate signal amplification or derivatization strategies, it is beneficial to further improve the detection sensitivity of the method so as to achieve the detection purpose. In this paper, according to the differences of ionization methods as well as research targets by mass spectrometry, the novel methods based on signal amplification strategy in recent ten years were classified and summarized. Considering of the current status with the status quo of ultra-sensitive detection by mass spectrometry, the future development prospect was also proposed.