In this study, the gas-assisted low-field magnetic separation (GAMS) was proposed for continuous large-scale magnetic bio-separation process. Magnetic recovery processes in selective separation of binary mixed proteins by magnetic microspheres were selected as a model. Basing on magnetic recovery efficiency of the batch GAMS for this model, the continuous GAMS with an amplifying small pilot scale was further researched. The continuous throughput of GAMS for adsorption, washing, and desorption step reached 12 L/h, 12 L/h, and 18 L/h with an adsorbent recovery above 95%, respectively. Furthermore, magnetic particles via the top magnetic roller could be well-separated from free proteins with good floatability via the bottom outlet of flotation column, and bubbling operation did not cause the significant denaturation of target protein. Compared with classic high gradient magnetic separation, GAMS had the advantages of easy scale-up, easy continuous operation, and low-cost, showing great potential for large-scale magnetic bio-separation process. (c) 2018 American Institute of Chemical Engineers AIChE J, 65: 175-183, 2019