Cervical cancer is a serious health problem in women. However, the use of clinical drug is seriously hampered by the development of drug resistance. Efficient in vitro tumor model is essential to improve the efficiency of drug screening and the accuracy of clinical application. Multicellular tumor spheroids (MTSs) can recapitulates tumor traits in vivo, thereby representing a powerful transitional model between 2D monolayer culture and xenograft. The cytotoxicity results showed the enhanced 5-fluorouracil (5-FU) resistance of HeLa carcinoma cells in 3D MTSs than 2D monolayer culture. This study formulates a rapid, scalable and reproducible in vitro model of 3D MTS for drug screening purposes, and the multi-OMICS analysis was made to better understand the key regulatory genes and regulated metabolic pathways responsible for the drug resistance mechanism.