We developed a localized single-cell electroporation chip to deliver exogenous biomolecules with high efficiency while maintaining high cell viability. In our microfluidic device, the cells are trapped in a microtrap array by flow, after which target molecules are supplied to the device and electrotransferred to the cells under electric pulses. The system provides the ability to monitor the electrotransfer of exogenous biomolecules in real time. We reveal through numerical simulations that localized electroporation is the mechanism of permeabilization in the microtrap array electroporation device. We demonstrate the simplicity and accuracy of this microtrap technology for electroporation by delivery of both small molecules using propidium iodide and large molecules using plasmid DNA for gene expression, illustrating the potential of this minimally invasive method to be widely used for precise intracellular delivery purposes (from bioprocess engineering to therapeutic applications).