Cancer cell separation is highly desirable for cancer diagnosis and therapy. Besides biochemical methods, engineered platforms are effective alternatives for sorting carcinoma cells from normal cells based on their unique properties in responding to the physical changes of the surrounding microenvironment. In this work, three-dimensional (3D) biomimetic scaffold platforms were developed to separate nasopharyngeal carcinoma 43 (NPC43) cells from immortalized nasopharyngeal epithelial 460 (NP460) cells based on precisely controlled design parameters including stiffness, number of layers, and structural layout. The migration characteristics of NPC43 and NP460 cells on the scaffold platforms revealed that NPC43 cells could squeeze into 10 µm wide, 15 µm deep trenches while NP460 cells could not. The different migration behavior was mainly due to cells having different interactions with the surrounding microenvironment. NPC43 cells had filopodia-like protrusions, while NP460 cells exhibited a sheet-like morphology. Using these 3D biomimetic platforms, 89% separation efficiency of NPC43 cells from NP460 cells was achieved on stiffer two-layer scaffold platforms with a 40/10 μm ridge/trench (R/T) grating on the top layer and a 20/10 μm R/T grid on the bottom layer. Moreover, the separation efficiency was further increased to 93% by adding an active conditioned medium (ACM) that caused the cells to have higher motility and deformability. These results demonstrate the capability to apply biomimetic engineered platforms with appropriate designs to separate cancer cells from normal cells for potential cancer diagnosis and treatment.