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Introduction: Cell separation is one of the important steps of purification in downstream processes. Some separation techniques such as centrifugation and filtration are expensive and would affect cell viability. Magnetic separation can be a good alternative for laboratory and industrial cell separation processes. Methods: For this purpose, L-lysine coated Iron Oxide Nanoparticles (IONs) were synthesized and used for magnetic separation of Escherichia coli as the most applied microbial cell in biotechnological processes. Results: IONs have successfully decorated the bacterial cells and cells were completely separated by applying an external magnetic field. Conclusion: This study showed that coating of E. coli cells with IONs could help to isolate cells from culture media without using expensive instruments.