13C metabolic flux analysis (13C-MFA) enables the precise quantification of intracellular metabolic reaction rates by analyzing the distribution of mass isotopomers of proteinogenic amino acids or intracellular metabolites through 13C labeling experiments. 13C-MFA has received much attention as it can help systematically understand cellular metabolic characteristics, guide metabolic engineering design and gain mechanistic insights into pathophysiology. This article reviews the advances of 13C-MFA in the past 30 years and discusses its potential and future perspective, with a focus on its application in industrial biotechnology and biomedicine.