In this study, we formulate a rapid, scalable and reproducible in vitro model of 3D MTS for drug screening purposes, and the cytotoxicity results showed an enhanced 5-FU resistance of HeLa carcinoma cells in 3D MTSs than 2D monolayer culture.In order to reveal the molecular mechanisms that drive 5-FU resistance in 3D HeLa carcinoma cells, a multi-omics study was applied to discover hidden biological regularities. We found that in the 3D MTSs mitochondrial function-related proteins and the metabolites of the tricarboxylic acid cycle were significantly decreased, and the cellular metabolism was shifted towards glycolysis. The differences in the protein synthesis, processing, and transportation between 2D monolayer cultures and 3D MTSs was significant, mainly in the heat shock protein family, with the upregulation of protein folding function in endoplasmic reticulum, which promoted the maintenance of ER homeostasis in the 3D MTSs. In addition, at the transcript and protein level, the expression of extracellular matrix proteins (e.g., laminin and collagen) were up-regulated in the 3D MTSs, which enhanced the physical barrier of drug penetration.