The microinjection of zebrafish larvae is a complicated work due to their small size and shape variations. In this article, for the first time, we design an automatic microinjection system dedicated to high-throughput injection of zebrafish larvae. The proposed custom-built microarray Petri dish combined with machine vision-based motion control enables high-efficiency automatic identification and injection of a batch of zebrafish larvae. The feasibility of the developed system is demonstrated by experimental testing on live zebrafish larvae. Results show that the developed system works fast with more stable injection success rate and higher survival rate than the skilled experimenter. The proposed system offers a good working efficiency and maintains a consistent success rate and stability in the process of high-throughput injection. It can be applied in various biomedical experiments based on zebrafish microinjection.