The 13C isotope tracer method was used to investigate the glucose metabolic flux distribution and regulation in Lactobacillus amylophilus to improve lactic acid production using kitchen waste saccharified solution (KWSS). The results demonstrate that L. amylophilus is a homofermentative bacterium. In synthetic medium, 60.6% of the glucose entered the Embden–Meyerhof–Parnas (EMP) to produce lactic acid, whereas 36.4% of the glucose entered the pentose phosphate metabolic pathway (HMP). After solid–liquid separation of the KWSS, the addition of Fe3+ during fermentation enhanced the NADPH production efficiency and increased the NADH content. The flux to the EMP was also effectively increased. Compared with the control (60.6% flux to EMP without Fe3+ addition), the flux to the EMP with the addition of Fe3+ (74.3%) increased by 23.8%. In the subsequent pyruvate metabolism, Fe3+ also increased lactate dehydrogenase activity, and inhibited alcohol dehydrogenase, pyruvate dehydrogenase and pyruvate carboxylase, thereby increasing the lactic acid production to 9.03 g l−1, an increase of 8% compared with the control. All other organic acid by-products were lower than in the control. However, the addition of Zn2+ showed an opposite effect, decreasing the lactic acid production. In conclusion it is feasible and effective means using GC-MS, isotope experiment and MATLAB software to integrate research the metabolic flux distribution of lactic acid bacteria, and the results provide the theoretical foundation for similar metabolic flux distribution.