Many transfection techniques can deliver biomolecules into cells, but the dose cannot be controlled precisely. Delivering well-defined amounts of materials into cells is important for various biological studies and therapeutic applications. Here, we show that nanochannel electroporation can deliver precise amounts of a variety of transfection agents into living cells. The device consists of two microchannels connected by a nanochannel. The cell to be transfected is positioned in one microchannel using optical tweezers, and the transfection agent is located in the second microchannel. Delivering a voltage pulse between the microchannels produces an intense electric field over a very small area on the cell membrane, allowing a precise amount of transfection agent to be electrophoretically driven through the nanochannel, the cell membrane and into the cell cytoplasm, without affecting cell viability. Dose control is achieved by adjusting the duration and number of pulses. The nanochannel electroporation device is expected to have high-throughput delivery applications.