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In this paper, we present a 3D printed tumor spheroidal model suitable for drug discovery. This model is based on a hydroxy-ethyl cellulose/alginate/gelatin (HCSG) composite biomaterial that has three distinct properties: (1) the HCSG is similar to the commercial basement membrane extract in Ki67, MUC1, and PARP1 expressions of MCF-7 cells for embedding culture; (2) the HCSG is printable at room temperature; and (3) the HCSG can be large-scale manufactured at an ultralow cost.We printed a 3D MCF-7 spheroid model with HCSG and characterized it in terms of cell viability, spheroid size, key protein expression, and mitochondrial metabolic activity. We used the 3D MCF-7 spheroid model to evaluate the anti-breast cancer activity of 13 amino acid-based flavone phosphoramidates and found that the alanine structure induced a stronger drug resistance, whereas phenylalanine hardly caused drug resistance in the MCF-7 cells. This is the first time that 3D bioprinting technology has been used in a structure–activity relationship study.