Performance of cross-flow ultrafiltration refolding strategy based on hollow fiber membrane for ribonuclease A was investigated. To improve the refolding efficiency, effects of operating conditions of ultrafiltration refolding on the activity yield and mass recovery were studied. Parameters of RNase A concentration (A), transmembrane pressure (B), circulation velocity (C) were chosen as the three test factors, with three level for each factor an L9(34) orthogonal array was implemented. Results of the orthogonal test showed that these three factors had a significant influence on the activity yield of the ultrafiltration refolding of ribonuclease A, whether based on variance analysis or range analysis. However, results of ANOVA (Analysis of Variance) showed that the three factors had no significant effect on mass recovery rate of ultrafiltration of ribonuclease A. Therefore, the optimal conditions for ultrafiltration of ribonuclease A were A1B1C2, that was, the concentration of refolding was 0.3 mg/mL, the transmembrane pressure was 34.0 kPa and the circulation velocity was 935 cm/min. Verification test was performed under the optimal conditions with the active yield of 92.31% and the mass recovery rate of 77.56%, respectively. Furthermore, the reliability of ultrafiltration refolding was confirmed by characterization of refolding products with circular dichroism, reversed-phase high performance liquid chromatography and high-performance gel filtration chromatography.