The Single-probe single-cell mass spectrometry (SCMS) is an innovative analytical technique designed for metabolomic profiling, offering a miniaturized, multifunctional device capable of direct coupling to mass spectrometers. It is an ambient technique leveraging microscale sampling and nanoelectrospray ionization (nanoESI), enabling the analysis of cells in their native environments without the need for extensive sample preparation. Due to its miniaturized design and versatility, this device allows for applications in diverse research areas, including single-cell metabolomics, quantification of target molecules in single cell, MS imaging (MSI) of tissue sections, and investigation of extracellular molecules in live single spheroids. This review explores recent advancements in Single-probe-based techniques and their applications, emphasizing their potential utility in advancing MS methodologies in microscale bioanalysis.