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Abstract: Biomedical field has been seeking a feasible standard drug screening system consisting of 3D tumor model array for drug researching due to providing sufficient samples and simulating actual in vivo tumor growth situation, which is still a challenge to rapidly and uniformly establish though. Here, we propose a novel drug screening system, namely 3D tumor array chip with “layer cake” structure, for drug screening. Accurate gelatin methacryloyl hydrogel droplets (~ 0.1 μL) containing tumor cells can be automatically deposited on demand with electrohydrodynamic 3D printing. Transparent conductive membrane is introduced as a chip basement for preventing charges accumulation during fabricating and convenient observing during screening. Culturing chambers formed by stainless steel and silicon interlayer is convenient to be assembled and recycled. As this chip is compatible with the existing 96-well culturing plate, the drug screening protocols could keep the same as convention. Important properties of this chip, namely printing stability, customizability, accuracy, microenvironment, tumor functionalization, are detailly examined. As a demonstration, it is applied for screening of epirubicin and paclitaxel with breast tumor cells to confirm the compatibility of the proposed screening system with the traditional screening methods. We believe this chip will potentially play a significant role in drug evaluation in the future.