Microinjection is an essential process in genetic engineering that is used to deliver genetic materials into various biological specimens. Considering the high-throughput requirement for microinjection applications ranging from gene editing to cell therapies, there is a need for an automated, highly parallelized, reproducible, and easy-to-use microinjection strategy. Here we report an on-chip, microfluidic microinjection module designed for compatibility with microfluidic large-scale integration (mLSI) technology and that can be fabricated via standard, multilayer soft lithography techniques. The needle-on-chip (NOC) module consists of a two-layer PDMS-based microfluidic module whose puncture and injection operations are reliant solely on Quake valve actuation. We designed a NOC module to conduct the microinjection of a common genetics model organism, Caenorhabditis elegans (C. elegans). The NOC design was analyzed using finite element method simulations for a large range of practically viable geometrical parameters. The computational results suggested that a slight lateral offset (>10 μm) of the control channel is sufficient for a successful NOC operation with a large fabrication tolerance (50 μm, 50% channel width). To demonstrate proof-of-concept, the microinjection platform was fabricated and utilized to perform a successful injection of a tracer dye into C. elegans.