Zebrafish (Danio rerio) has recently emerged as a powerful experimental model in drug discovery and environmental toxicology. Notwithstanding this, automated in-situ analysis of zebrafish embryos is still deeply in its infancy. Currently available technologies do not allow for an automated loading, trapping, addresses designation to each embryo during analysis and perfusion treatment of large numbers of immobilized zebrafish embryos. This work describes the proof-of-concept design, development and validation of a 3D multilayer microfluidic chip-based system for real-time developmental analysis of zebrafish embryos. The design achieves one-embryo-in-one-trap for convenient address designation and encoding to each embryo and is capable of high-throughput docking and recovery of single embryos at a large scale. The chip-based design features integrated heating manifolds and is interfaced with a piezo-electric ultrasonic micro-pump for efficient chip actuation.