Metabolic flux analysis using 13C enrichment data of intracellular free amino acids (FAAs) can improve the time resolution of flux estimation compared to analysis of proteinogenic amino acid data owing to the faster turnover times of FAAs. The nature of the 13C enrichment dynamics of FAAs remains obscure, however, especially with regard to its dependence on culture conditions, even though an understanding of dynamic behavior is important for precise metabolic flux estimation. In this study, we analyzed the 13C enrichment dynamics of free and proteinogenic amino acids in a series of continuous culture experiments with Escherichia coli. The results indicated that the effect of protein degradation on the 13C enrichment of FAAs was negligible under cellular growth conditions. Furthermore, they showed that the time scale necessary for 13C enrichment dynamics of FAAs to reach a steady state depends on culture conditions such as oxygen uptake rate, which was likely due to different pool sizes of intracellular metabolites. The results demonstrate the importance of analyzing 13C enrichment dynamics for the precise estimation of metabolic fluxes using FAA data.