Abstract: As the fundamental units of the structure and function of living organisms, cells perform the core functions of life activities. With the deepening research in life sciences, the importance of studies on cellular heterogeneity has become increasingly prominent. Consequently, the accurate analysis of single cells has attracted growing attention. However, single cells are extremely small in volume, and their internal components are not only complex but also present in extremely low concentrations. Furthermore, mutual interference occurs between different substances during the analysis process. Together, these factors pose immense challenges to single-cell analysis. Mass spectrometry (MS), boasting versatility, label-free detection, high sensitivity, high resolution, and excellent selectivity, has emerged as a highly promising analytical tool for single-cell studies. Compared with conventional mass spectrometry, surface desorption ambient mass spectrometry (SDAMS) exhibits distinct advantages that are tailored to single-cell analysis. Specifically, SDAMS enables direct sample analysis under ambient pressure, eliminating the need for a vacuum system and complex sample pretreatment steps. Furthermore, SDAMS facilitates in situ real-time detection with high spatial resolution. These attributes make SDAMS particularly well-suited for analyzing trace and complex systems, such as single cells, especially those that grow adherently or are attached to surfaces. This review systematically summarized recent progress in SDAMS-based single-cell analysis, focusing on five representative techniques: 1) Desorption electrospray ionization mass spectrometry (DESI-MS) and its subtype, Nano-DESI-MS; 2) Electrospray ionization-derivatization mass spectrometry, which includes paper spray ionization mass spectrometry (PSI-MS) and glass electrospray ionization mass spectrometry (GESI-MS); 3) Probe electrospray ionization mass spectrometry (PESI-MS); 4) Laser-assisted electrospray ionization mass spectrometry (encompassing laser ablation electrospray ionization mass spectrometry (LAESI-MS) and atmospheric pressure matrix-assisted laser desorption ionization mass spectrometry (AP-MALDI-MS); 5) Dielectric barrier discharge ionization mass spectrometry (DBDI-MS). These techniques provide versatile platforms for investigating intracellular metabolites, proteins, and small molecules in their native state, thereby facilitating breakthroughs in fields such as cancer biology, neuroscience, and drug discovery. Nevertheless, this class of technologies still suffers from unresolved limitations, including insufficient spatial resolution for subcellular-level analysis, trade-offs between sensitivity and analytical throughput, and potential sample damage. To address these challenges, this review outlined key future development directions, with a primary emphasis on the integration of multiple technologies and instrumental optimization. Overall, this review highlighted the immense potential of SDAMS to revolutionize single-cell analysis and provided insights for overcoming technical bottlenecks to fully unlock its clinical and research value.