Abstract: AbstractEsophageal cancer (EC) is characterized by high morbidity and mortality, and chemotherapy has become an indispensable means for comprehensive treatment. However, due to the limitation of the effective in vitro disease model, the development of chemotherapeutic agents still faces great challenges. In this paper, we present a novel tumor spheroid on a chip platform based on inverse opal hydrogel scaffolds to screen chemotherapeutic agents for EC treatment. With the microfluidic emulsion approach, the inverse opal hydrogel scaffolds were generated with tunable and organized pores, which could provide spatial confinement for cell growth. Thus, the suspended KYSE-70 cells could successfully form uniform cell spheroids on the inverse opal hydrogel scaffolds. It was demonstrated that the tumor cell spheroids could recapitulate 3D growth patterns in vivo and exhibited higher sensitivity to the chemotherapy agents compared with monolayer cells. Besides, by employing the scaffolds into a microfluidics to construct esophageal tumor on a chip, the device could realize high-throughput tumor cell spheroids generation and drug screening, indicating its promising role in chemotherapy drug development.