Understanding the physiologies and pathologies of diseases requires a thorough understanding of metabolic heterogeneity in cells. This technical note presents a 3D printing technology for manufacturing an ionization source that is specially adapted for mass spectrometry-based single-cell analysis. This all-in-one 3D-printed electrospray ionization source integrates the sample introduction, metabolite extraction, and ionization into one device, simplifying the process of single-cell analysis and improving the reproducibility of the measurement. We successfully used it for high-throughput analysis of three types of cancer cells (around 17 cells/min) and used the t-distributed stochastic neighbor embedding algorithm to distinguish different cell types based on detected metabolites. By simply adjusting the printing parameters of the 3D-printed ionization source, it can be applied to cells with different sizes. The proposed 3D-printed ionization source promises to open new possibilities for single-cell analysis.