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Liver transplantation is currently the sole definitive treatment option for end‐stage liver failure. However, a significant shortage of donors prevails due to high clinical demands. Recently, human liver organoids have shown significant potential in regenerative medicine for liver diseases. Nevertheless, current static cultures of organoids grown in well‐plates heavily rely on extracellular matrix hydrogels (Matrigel), thereby limiting both the scalability and quantity of organoid culture. In this study, we present a groundbreaking culture mode that eliminates all reliance on extracellular matrix hydrogels, enabling the successful preparation of functional human liver ductal organoids (LDOs) based on the cell suspension culture mode in a mechanically stirred bioreactor. Initially, the developed suspension culture in a 6‐well plate without matrigel was proven to support robust growth of liver ductal organoids with an average size 2.6 times larger than those obtained in static culture, and with a high organoid survival rate exceeding 90%. Also, the transcriptome profile reveals that suspension culture activates the phosphatidylinositol 3‐kinase (PI3K) signalling pathway through mechanical signal transduction, thereby promoting hepatobiliary characteristics. Then, a controllable and scalable bioprocess for liver ductal organoid culture was developed and successfully scaled up to a 50 mL flask bioreactor with a working volume of 15 mL. Finally, animal experiments indicated that the transplantation of liver ductal organoids harvested from suspension culture can effectively alleviate liver injury and inflammation, demonstrating the feasibility of large‐scale production of liver ductal organoids cultivated in suspension culture with an improved extracellular matrix environment. A novel matrigel‐free suspension culture enhances the generation of high‐quality human liver ductal organoids, which shows remarkable efficacy in mice transplantation.