Statement of Purpose: Hydrogel multilayers with micrometer-scale in thickness containing living cancer cells were prepared by layer-by-layer assembling with 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer solution and poly(vinyl alcohol) (PVA) solution alternatively under biological conditions. It was demonstrated that this tool could be utilized for minimizing barriers to the diffusion of drugs while preserving the 3D context. As a case study, the cytotoxicity effects of paclitaxel (PTX) on human cervical cancer HeLa-Fucci cells were evaluated in vitro. A comparative study was carried out: 2D culture, 3D culture in microgel thin membrane and in macrogel. Time-lapse microscopic analyses showed HeLa-Fucci cells cultured on 2D exhibited a more rapid and robust response to stimulation than cells cultured in microgel thin membrane. Besides, HeLa-Fucci cells cultured under macrogel did not show any response to stimulation. It was speculated that both of the diffusion of PTX and matrix dimensionality could affect drug efficacy and diffusion of drug seemed to play a more important role.