Retinal organoids (ROs) derived from human pluripotent stem cells (hPSCs) have become a promising model in vitro to recapitulate human retinal development, which can be further employed to explore the mechanisms of retinal diseases. However, the current culture systems for ROs lack physiologically relevant microenvironments, such as controllable mechano-physiological cues and dynamic feedback between cells and the extracellular matrix (ECM), which limits the accurate control of RO development. Therefore, we designed a controllable perfusion microfluidic chip (CPMC) with the advantages of precisely controlling fluidic shear stress (FSS) and oxygen concentration distribution in a human embryonic stem cell (hESC)-derived RO culture system. We found that ROs cultured under this system allow for expanding the retinal progenitor cell (RPC) pool, orchestrating the retinal ganglion cell (RGC) specification, and axon growth without disturbing the spatial and temporal patterning events at the early stage of RO development. Furthermore, RNA sequencing data revealed that the activation of voltage-gated ion channels and the increased expression of ECM components synergistically improve the growth of ROs and facilitate the differentiation of RGCs. This study elaborates on the advantages of the designed CPMC to promote RO growth and provide a controllable and reliable platform for the efficient maturity of RGCs in the ROs, promising applications in modeling RGC-related disorders, drug screening, and cell transplantation.