Intracellular delivery of functional molecules such as proteins, transcription factors and DNA is effective and promising in cell biology. However, existing transfection methods are often unsuitable to deliver big molecules into cells or require carriers such as viruses and peptides specific to the target molecules. In addition, the nature of bulk processing does not generally provide accurate dose control of individual cells. The concept of single-cell-based material injection based on electrokinetic pumping through nanocapillaries could overcome these problems, yet the fabrication and operation of nanoscale 3-dimensional structures have remained unsolved. In this research, a hybrid (PDMS/glass) microfluidic chip with a true 3-dimensional nanoinjection structure (called "nanoinjection system") is presented. The nanoinjection structure was fabricated by femtosecond-laser (fs-laser) ablation in a single solid glass, which showed very successful delivery of red fluorescent protein (RFP) and expression of plasmid DNA in several different types of cells. This system is promising in that the amount of molecules to be delivered is controllable and the processed cells are systematically separated into a harvesting chamber, which can radically improve the purity of the processed cells. In addition, it was confirmed that the cells were healthy even after the molecule injection for a few seconds, indicating that the injection time can be significantly elongated, further improving the delivery efficiency of biomolecules without affecting the cell viability. We envision that the nanoinjection system having the major features of being carrier-free and dose-controllable, having an unlimited injection period, and ease of harvesting will greatly contribute to the next-generation research studies in the fields of cell biology and cell therapeutics.