This study describes a novel method for single-cell analysis and lipid profiling by combining drop-on-demand inkjet cell printing and probe electrospray ionization mass spectrometry (PESI-MS). Through inkjet sampling of a cell suspension, droplets with single cells were generated, precisely dripped onto a tungsten-made electrospray ionization needle, and immediately sprayed under a high-voltage electric field. Lipid fingerprints of single cells were obtained by a mass spectrometry (MS) detector. A homemade magnetic stirring device was applied to the cell suspension reservoir, which controlled the homogeneous distribution of cells in liquid and improved the single-cell-droplet percentage by 43.8%. Eight types of single cells were screened in our platform and further differentiated by principal component analysis based on cellular surface phospholipids. Thus, this study successfully provides a facile method for the direct MS profiling of single-cell lipids by PESI-MS.