Protein refolding is still a bottleneck for large-scale production of valuable proteins expressed as inclusion bodies in Escherichia coli. Usually biologically active proteins cannot be obtained with high yield at a high concentration after refolding. In order to meet the challenge of protein refolding a urea gradient gel filtration-refolding system was developed in this article. A Superdex 75 column was pre-equilibrated with a linear decreased urea gradient, the denatured protein experienced the gradual decrease in urea concentration as it went through the column. The refolding of denatured lysozyme showed this method could significantly increase the activity recovery of denatured lysozyme at high protein concentration. The activity recovery of 90% was obtained from the initial protein concentration up to 17 mg/ml within 40 min.