Development of an in vitro three-dimensional (3D) model that closely mimics actual environment of tissue has become extraordinarily important for anti-cancer study. In recent years, various 3D cell culture systems have been developed, with multicellular tumor spheroids being the most popular and effective model. In this work, we present a microfluidic device used as a robust platform for generating core–shell hydrogel microspheres with precisely controlled sizes and varied components of hydrogel matrix. To gain a better understanding of the goveing mechanism of microsphere formation, computational models based on multiphase flow were developed to numerically model the droplet generation and velocity field evolution process with COMSOL Multiphysics software. Our modeling results show good agreement with experiments in size dependence on flow rate as well as effect of vortex flow on microsphere formation. With real-time tuning of the flow rates of aqueous phase and oil phase, tumor cells were encapsulated into the microspheres with controllable core–shell structure and different volume ratios of core (comprised of alginate, Matrigel, and/or Collagen) and shell (comprised of alginate). Viability of cells in four different hydrogel matrices were evaluated by standard acridine orange (AO) and propidium iodide (PI) staining. The proposed microfl uidic system can play an important role in engineering the in vitro micro-environment of tumor spheroids to better mimic the actual in vivo 3D spatial structure of a tumor and perfect the 3D tumor models for more effective clinical therapies.