Three-dimensional (3D) hydrogel microspheres have attracted increasing attention as cell culture carriers. The system of hydrogel microspheres provides great advantages for cell growth owing to its high surface-to-volume ratio and biocompatible environment. However, an integrated system that includes microsphere generation, microsphere capture and in situ culture together has not been realized yet. Here we present a multifunctional microfluidic device to accomplish the overall process including cell-laden microsphere generation, online demulsification and dynamic-culture. The microfluidic device can produce massive monodispersed alginate microspheres and allows us to immobilize the alginate microspheres and record bacterial growth. Moreover, the microspheres provide a suitable environment through the mechanical properties of soft tissues, leading to high cell viability, proliferation, activity and biocompatibility. We believe that this versatile and biocompatible platform will provide a more reliable analysis tool for tissue engineering and cell therapy.