The development and application of miniaturized platforms with the capability for microscale and dynamic control of biomimetic and high-throughput three-dimensional (3D) culture plays a crucial role in biological research. In this study, pneumatic microstructure-based microfluidics was used to systematically demonstrate 3D tumor culture under various culture conditions. We also demonstrated the reusability of the fabrication-optimized pneumatic device for high-throughput cell manipulation and 3D tumor culture. This microfluidic system provides remarkably long-term (over 1 month) and cyclic stability. Furthermore, temporal and high-throughput monitoring of tumor response to evaluate the therapeutic efficacy of different chemotherapies, was achieved based on the robust culture. This advancement in microfluidics has potential applications in the fields of tissue engineering, tumor biology, and clinical medicine; it also provides new insight into the construction of high-performance and recyclable microplatforms for cancer research.