The invention discloses a concentration gradient generation device for gel 3D cell culture, which includes a multi-stage mixing and concentration gradient generation module, and a gel chamber with a multi-stage concentration gradient range, which can simultaneously load multiple gel samples , and through the application of segmental concentration gradients, the detection and evaluation of cell behavior in gels in different concentration gradient ranges is carried out at the same time; the establishment of multi-level concentration gradient ranges can simultaneously detect cells in different concentrations on the same fluid chip. span samples; the fluid chip provided by the present invention has the advantages of small size, low cost, and good integration performance with microgels, and has broad applications in in vitro tissue reconstruction, cell physiology, pathology, and drug screening, biomedicine, and environmental monitoring. Application prospect; at the same time, the invention also discloses its preparation method and application method.