Background Endotoxin is a major process-related impurity that can act as a strong immunostimulant leading to fever and hypotensive shock. Thus, the US FDA and international quality standards strictly direct the biologics manufacturers to control the endotoxin contamination during the purification process. In this work, a developed method for biologics purification from acquired endotoxin contamination is introduced. This is accomplished by the preparation of dextran-coated magnetic nanoparticles using a facile rapid co-precipitation method. Results The resulting magnetic nanoparticles (MNPs) are characterized by dynamic light scattering, transmission electron microscope, Fourier transform infrared spectroscopy, X-ray diffraction, and vibrating sample magnetometry. The dextran-coated magnetic nanoparticles are further coupled to either polymyxin B or histidine to provide a positively charged ligand which enhances the affinity to the negatively charged endotoxin. Both ligands-coupled MNPs are tested for purification efficiency using the chromogenic kinetic assay. The method conditions are optimized using a two-level factorial design to achieve best purification conditions of the contaminated biologics and indicated endotoxin removal percentage 85.12% and maximum adsorption capacity of 38.5 mg/g, for histidine-coupled MNPs. Conclusions This developed method is introduced to serve biologics manufacturers to improve their manufacturing processes through providing a simple purifying tool for biologics from acquired endotoxin contamination. Graphical Abstract