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Abstract: Traditional tumor models do not tend to accurately simulate tumor growth in vitro or enable personalized treatment and are particularly unable to discover more beneficial targeted drugs. To address this, this study describes the use of threedimensional (3D) bioprinting technology to construct a 3D model with human hepatocarcinoma SMMC-7721 cells (3DP- 7721) by combining gelatin methacrylate (GelMA) and poly(ethylene oxide) (PEO) as two immiscible aqueous phases to form a bioink and innovatively applying fluorescent carbon quantum dots for long-term tracking of cells. The GelMA (10%, mass fraction) and PEO (1.6%, mass fraction) hydrogel with 3:1 volume ratio offered distinct pore-forming characteristics, satisfactory mechanical properties, and biocompatibility for the creation of the 3DP-7721 model. Immunofluorescence analysis and quantitative real-time fluorescence polymerase chain reaction (PCR) were used to evaluate the biological properties of the model. Compared with the two-dimensional culture cell model (2D-7721) and the 3D mixed culture cell model (3DM-7721), 3DP-7721 significantly improved the proliferation of cells and expression of tumor-related proteins and genes. Moreover, we evaluated the differences between the three culture models and the effectiveness of antitumor drugs in the three models and discovered that the efficacy of antitumor drugs varied because of significant differences in resistance proteins and genes between the three models. In addition, the comparison of tumor formation in the three models found that the cells cultured by the 3DP-7721 model had strong tumorigenicity in nude mice. Immunohistochemical evaluation of the levels of biochemical indicators related to the formation of solid tumors showed that the 3DP-7721 model group exhibited pathological characteristics of malignant tumors, the generated solid tumors were similar to actual tumors, and the deterioration was higher. This research therefore acts as a foundation for the application of 3DP-7721 models in drug development research.