Based on optimization of denaturing conditions and the character of time course of protein refolding, renaturation by dilution in batch and fed-batch operation to improve yield as well as the initial and final protein concentrations has been studied. The optimum DTT in denatured solution was 30 mM. Urea can suppress protein aggregation to sustain pathway of correct refolding at high protein concentration. Fed-batch operation was better than batch dilution with comparison of yield recovery in large-scale downstream processes. Under our research condition in fed-batch operation, lysozyme was successfully refolded from initial protein concentration of up to 40 mg/mL by dilution 20-fold, the yield recovery was nearly 60%.