This study of renaturation by dilution and size exclusion chromatography (SEC) addition of urea to improve yield as well as the initial and final protein concentrations showed that although urea decreased the rate of lysozyme refolding, it could suppress protein aggregation to sustain the pathway of correct refolding at high protein concentration; and that there existed an optimum urea concentration in renaturation buffer. Under the above conditions, lysozyme was successfully refolded from initial concentration of up to 40 mg/mL by dilution and 100 mg/mL by SEC, with the yield of the former being more than 40% and that of the latter being 34.8%. Especially, under the condition of 30 min interval time, i.e. τ>2(tR2-tR1), the efficiency was increased by 25% and the renaturation buffer could be recycled for SEC refolding in continuous operation of downstream process.