Abstract: This paper aims to explore the conditions of gene transfection utilizing the three-dimensional(3D)cell culture model.Firstly,3D cell model was established by vitro using semisolid culture system of methyl cellulose.Then Cy5 labeled gene and enhanced green fluorescence protein(EGFP)report gene were transfected into 3D cell model via polyethylene imine(PEI,25 kDa).The gene delivery and EGFP expression was analyzed through 3D reconstruction method using confocal laser scanning microscope.The results showed that when the composite ratio of PEI to plasmid DNA was 10,the PEI-mediated gene had good transport ability and certain expression ability in the 3D cell model.The results of this study can provide experimental basis for the application of 3D cell model in gene transfection.