Magnetic separation is a promising alternative to conventional methods in downstream processing. This can facilitate easier handling, fewer processing steps, and more sustainable processes. Target materials can be extracted directly from crude cell lysates in a single step by magnetic nanoadsorbents with high-gradient magnetic fishing (HGMF). Additionally, the use of hazardous consumables for reducing downstream processing steps can be avoided. Here, we present proof of principle of one-step magnetic fishing from crude Escherichia coli cell lysate of a green fluorescent protein (GFP) with an attached hexahistidine (His6)-tag, which is used as the model target molecule. The focus of this investigation is the upscale to a liter scale magnetic fishing process in which a purity of 91% GFP can be achieved in a single purification step from cleared cell lysate. The binding through the His6-tag can be demonstrated, since no significant binding of nontagged GFP toward bare iron oxide nanoparticles (BIONs) can be observed. Nonfunctionalized BIONs with primary particle diameters of around 12 nm, as used in the process, can be produced with a simple and low-cost coprecipitation synthesis. Thus, HGMF with BIONs might pave the way for a new and greener era of downstream processing.