Nasopharyngeal carcinoma (NPC) is a prominent head and neck malignancy, yet the mechanisms underlying its occurrence, progression, recurrence, metastasis, drug resistance, and radiation resistance have not been fully understood. This knowledge gap is partly due to the lack of preclinical models of NPC research. Compared to traditional 2D cell cultures, 3D bioprinting (3DP) offers significant advantages in replicating the tumor microenvironment. However, no studies to date have used 3D bioprinting technology to model NPC. In this study, we used extrusion-based 3D bioprinting to develop a new preclinical NPC model (3DP-HK1) using the emerging bio-ink gelatin methacryloyl (GelMA). The model successfully demonstrated the ability to sustain long-term tumor cell activity. Immunohistochemistry and immunofluorescence analyses demonstrated that 3DP-HK1 largely retained the histopathological features and tumor-related protein expression of NPC. In addition, we conducted a wound healing experiment, which indicated that tumor cells in 3DP-HK1 have stronger migration ability than 2D-cultured cells (2D-HK1), highlighting differences in cellular phenotype. The different responses of 3DP-HK1 and 2D-HK1 to various anti-tumor drugs and radiation reflect the advantages of 3DP-HK1 for preclinical drug screening and exploring mechanisms of radiotherapy in NPC. Transcriptome sequencing revealed that 3DP-HK1 has a distinct gene expression profile compared to 2D-HK1, with significantly upregulated expression of malignant genes, such as keratin 6B (KRT6B), S100 calcium binding protein A8 (S100A8), and crystallin alpha B (CRYAB). Meanwhile, genes associated with drug resistance (e.g., lysine demethylase 5B [KDM5B]) and radiation resistance (e.g., carnitine palmitoyltransferase 1A [CPT1A]) were also upregulated, confirming findings from other experimental analyses at the RNA level. In conclusion, this study successfully constructed a 3D bioprinting-based preclinical model for NPC research and proved its reliability and significant potential for advancing drug screening and mechanistic studies.