We report a concentration gradient generator featuring valve-free and simultaneous two-dimensional (2D) concentration generation. Each analyte is first diluted by a 1D gradient generator to five different concentrations and then directed into different chambers to generate combinations of different concentrations. The chip consists of three layers, which realizes a 3D fluid path, forming a skywalk structure that allows two perpendicular channels to cross each other in different layers. In this way, this chip could get rid of the pneumatic actuated valves (PAVs) or complicated channel designs that are conventionally adopted in controlling the infusion of multi-analytes. We believe that this design, which makes the chip independent of cumbersome external apparatus in controlling multi-analytes infusion, may potentially benefit the process of making microfluidic chips a portable and cost-effective product.