Three-dimensional culture of tumor cell spheres can truly simulate the growth environment of tumor cells in vivo, and thus be more suitable for drug screening and other related research. Combined with microfluidic technology, there are more ways to implement three-dimensional cell culture technology. In this study, a microfluidic chip for high-throughput three-dimensional tumor cell spheres culture was designed to enable rapid cultivation and drug screening of tumor cell spheres based on microcavity arrays. Compared to the current mainstream three-dimensional culture that takes 4 days, microfluidic chips only require 15 h to form tumor spheres. It significantly saves cultivation time. The reliability of three-dimensional culture in the chip was verified by tumor growth morphology monitoring and fluorescence imaging technology. Meanwhile, this research realized multi-drug rapid screening based on chip, presented good gradient dependence and verified the feasibility of drug screening in chip. This high-throughput microfluidic chip can be applied in multiple research fields such as oncology, providing a new technological means and experimental platform for achieving rapid drug screening in tumors.