Cancer immunotherapy is greatly advanced by the effective synthesis of therapeutic proteins, such as single-chain variable fragment (scFv) antibodies. This study assesses and contrasts the functionality and production of Anti-PD-L1 scFv-Fc antibodies expressed in HEK293 cells and Escherichia coli (E. coli) strains (Rosetta (DE3) and SHuffle^®). Each platform was optimized by systematically evaluating key parameters such as biological activity, protein solubility, and expression yield. By adjusting the temperature, IPTG concentration, and induction duration, expression in E. coli was optimized. SHuffle^® achieved superior protein folding and binding compared to Rosetta (DE3), attributed to enhanced disulfide bond formation. Flow cytometry assays using PD-L1-positive MDA-MB-231 cells demonstrated that Anti-PD-L1 scFv-Fc produced in HEK293 cells exhibited superior binding specificity compared to proteins expressed in E. coli SHuffle^® and Rosetta (DE3) strains. Although ELISA confirmed specific binding of scFv-Fc to PD-L1 for HEK293 and SHuffle^® samples, the differences were not statistically significant. Most functionally active scFv-Fc was produced by HEK293 cells, but SHuffle^® provided a less expensive substitute with similar binding capabilities. The therapeutic potential of the antibodies was highlighted by flow cytometry, which verified the selective binding of scFv-Fc to PD-L1-positive cells with minimal activity against PD-L1-negative controls. In conclusion, HEK293 cells continue to be the recommended option for applications needing high antigen-binding, but SHuffle^® offers a practical, affordable platform for creating functionally active Anti- PD-L1 scFv-Fc antibodies. The results of this study will aid in the development of potent therapeutic agents for immune checkpoint blockade therapy and optimize scFv-Fc production strategies.