In this study, we developed a novel three-dimensional (3D) cancer cell chip using a three-floor hierarchical 3D pyramid structure (3D pyramid) to simulate 3D tumor cell growth in vitro and to detect anticancer drugs. The proposed 3D pyramidbased cancer cell chip offered substantial advantages for the agglomerate formation of tumor cells, in which cells could be maintained as tumor spheroids for up to 3 weeks. Soon after HeLa tumor cells adhered to the micropatterned pillar sidewalls, they were suspended between the pillars based on scanning electron microscopy images. Treatment with the anticancer drug oleanolic acid resulted in 46.33% and 5.86% apoptotic cells on the 2D plate and 3D pyramid-based cell chip, respectively, compared with only 0.06% apoptotic cells in the control. The increase in chemoresistance to anticancer drugs in the 3D pyramid-based cell chip might be a result of cell confluence and hypoxia due to the spheroid formation of tumor cells in the 3D pyramid structure. These results indicated that the proposed cell chip could potentially be used for anticancer drug screening or can be incorporated into other models aimed at prolonging various cell functions in culture.