Establishing a physiological microenvironment in vitro that is suitable for cell and tissue growth is essential for medical research. Microfluidic chips are widely used in the construction of a microenvironment and the analysis of cell behavior in vitro; however, the design and manufacture of microfluidic chips for the long-term culture of a tumor model tends to be highly complex and time-consuming. In this paper, we propose a method for the rapid fabrication of a microfluidic chip for multi-dimensional cell co-culture. A major advantage of this method is that the microfluidic chip can be divided into several sections by micro-pillar arrays to form different functional regions to grow two- and three-dimensional cell culture on the same matrix. At the micro-scale, the surface tension between the gelatin methacryloyl-encapsulated cells and micro-pillars prevents the leakage of the hydrogel, and the hydrogel provides a three-dimensional microenvironment for cell growth. Our results of long-term cell culture and preclinical drug screening showed that cells cultured in a two-dimensional monolayer differ from three-dimensional cultured cells in terms of morphology, area, survival rate, proliferation, and drug resistance. This method shows potential for use in the study of cell behavior, drug screening, and tissue engineering.