Patients with gastric cancer exhibit considerable genetic and phenotypic heterogeneity, necessitating the establishment of a model for personalized medicine screening. Here, we successfully established three-dimensional cell spheroids (3D cell spheroids) from fresh gastric cancer tissues (n = 30) as in vitro models for further therapeutic screening. Hematoxylin-eosin and immunohistochemical staining of whole spheroids and parental tumor tissues revealed that the 3D cell spheroids recapitulated the parental tissue structure and maintained the histological characteristics of the parental tumor tissue during long-term expansion in vitro. Further, transcriptome sequencing verified that the cell spheroids could recapitulate the gene expression profile characteristics of the parental tumor tissue. Drug susceptibility testing of the 3D cell spheroids demonstrated that these cell spheroids can be used as a reliable model for drug prediction.