The current state-of-art in 3D microfluidic chemotaxis device (microFCD) is limited by the inherent coupling of the fluid flow and chemical concentration gradients. Here, we present an agarose-based 3D microFCD that decouples these two important parameters, in that the flow control channels are separated from the cell compartment by an agarose gel wall. This decoupling is enabled by the transport property of the agarose gel, which-in contrast to the conventional microfabrication material such as polydimethylsiloxane (PDMS)-provides an adequate physical barrier for convective fluid flow while at the same time readily allowing protein diffusion. We demonstrate that in this device, a gradient can be pre-established in an agarose layer above the cell compartment (a gradient buffer) before adding the 3D cell-containing matrix, and the dextran (10 kDa) concentration gradients can be re-established within 10 min across the cell-containing matrix and remain stable indefinitely. We successfully quantified the chemotactic response of murine dendritic cells to a gradient of CCL19, an 8.8 kDa lymphoid chemokine, within a type I collagen matrix. This model system is easy to set up, highly reproducible, and will benefit research on 3D chemoinvasion studies, for example with cancer cells or immune cells. Because of its gradient buffering capacity, it is particularly suitable for studying rapidly migrating cells like mature dendritic cells and neutrophils.