Recombinant Escherichia coli BL21 was used to produce human-like collagen II in fed-batch cultivation. By performing MFA (metabolic-flux analysis), the carbon/nitrogen molar ratios in both the batch and feeding media were optimized for high-level production of human-like collagen II. Three carbon/nitrogen molar ratios in both the batch and feeding media were used in the present study, and the MFA results showed that the optimal carbon/nitrogen molar ratios for the batch and feeding media were 2.36:1 and 5.12:1 respectively, yielding the highest dry-cell density (67.2 g/l dry cell weight) and human-like collagen production (10.8 g/l).