In previous years, three-dimensional (3D) cell culture technology has become a focus of research in tumor cell biology, using a variety of methods and materials to mimic the in vivo microenvironment of cultured tumor cells ex vivo. These 3D tumor cells have demonstrated numerous different characteristics compared with traditional two-dimensional (2D) culture. 3D cell culture provides a useful platform for further identifying the biological characteristics of tumor cells, particularly in the drug sensitivity area of the key points of translational medicine. It promises to be a bridge between traditional 2D culture and animal experiments, and is of great importance for further research in the field of tumor biology. In the present review, previous 3D cell culture applications, focusing on anti-tumor drug susceptibility testing, are summarized.