The invention discloses a three-dimensional cell co-culture model based on PDMS (polydimethylsiloxane). The three-dimensional cell co-culture model comprises a model body, wherein a left culture pool and a right culture pool are arranged on the inner side of the model body, an interlayer is arranged between the left culture pool and the right culture pool, an opening communicating the left culture pool with the right culture pool is formed in the interlayer, and stoppers jointed with the side face of the interlayer are arranged in the left culture pool and the right culture pool. According to the three-dimensional cell co-culture model based on PDMS, a co-culture system of two kinds of cells is established in the same plane so as to dynamically observe the interaction of the two kinds of cells, cell migration, chemotaxis, invasion and tube formation tests can be carried out, molecular biology detection can be conveniently carried out on the cells in situ or the cells are connected to perform the detection, and in addition, the defect that a vertical co-culture method cannot eliminate gravity interference is overcome; and the three-dimensional cell co-culture model can be repeatedly used and has the advantages of being simple and practicable, good in repeatability, cheap, safe and the like. The invention further discloses a preparation method of the model. The preparation method is simple in technique, liable to operate and easy to control.