Abstract: Human embryonic kidney 293 (HEK293) cell line has become a widely employed platform for recombinant protein production, owing to its human origin, ease of transfection, and adaptability to various expression systems. Its transient expression system is particularly advantageous for the rapid synthesis of proteins, making it well-suited for short-term experimental applications. However, this method is limited in scalability, primarily due to the high costs associated with large-scale plasmid preparation and transfection reagents. In contrast, stable expression systems-achieved through genomic integration of the target gene-enable continuous and reproducible protein production. Nevertheless, the efficiency of stable expression in HEK293 cells is typically lower than that in Chinese hamster ovary (CHO) cells, particularly in critical processes such as gene screening, clonal selection, and the maintenance of long-term expression stability. Noggin, a secreted protein that plays a pivotal role in regulating organoid development and differentiation, is commonly produced via transient transfection in HEK293 cells. This approach frequently results in substantial batch-to-batch variability, which can undermine reproducibility in downstream applications. To address this limitation, a stable HEK293 cell line expressing Noggin protein was developed through gene transfection and screening protocols.Subsequently, the culture conditions were optimized to support high-level expression. Noggin protein was purified to high purity levels using a combination of affinity chromatography and cation-exchange chromatography. Experimental data revealed that compared with the transient expression system, the stable expression system increased protein yield by approximately 15-fold. The biological activity of the purified Noggin protein was confirmed through functional assays using murine small intestinal crypt organoids, thereby verifying its effectiveness in supporting organoid growth. This stable expression system not only improves production yield, but also ensures consistent protein quality. It provides a reliable and scalable solution for Noggin protein production, thus facilitating its application in basic research.