Concentration gradients play an important role in controlling biological and pathological processes, such as metastasis, embryogenesis, axon guidance, and wound healing. Microfluidic devices fabricated by photo- and soft lithography techniques can manipulate the fluidic flow and diffusion profile to create biomolecular gradients in a temporal and spatial manner. Furthermore, microfluidic devices enable the control of cell-extracellular microenvironment interactions, including cell cell, cell matrix, and cell soluble factor interaction. In this paper, we review the development of microfluidic-based gradient devices and highlight their biological applications.