[Objective] The mechanism of L-threonine biosynthesis and its impact factors were explored by metabolic flux analysis. [Methods] The metabolic flux balance model of L-threonine synthesis by Escherichia coli was established. Based on this model, the practical and optimal metabolic flux distribution in the middle and late period under different dissolved oxygen concentrations were determined with the linear program planted in MATLAB software. [Results] Data indicated that 25.5% of carbon sources were consumed by HMP pathway, resulting in a conversion rate of 33.9% to L-threonine with a 5% dissolved oxygen concentration. With dissolved oxygen concentration of 20%, 58.08% carbon resources entered HMP pathway, giving rise to a 46.5% conversion rate. [Conclusion] Compared to the optimal metabolic flux with a carbon conversion rate of 88.23%, glucose-6-phosphate isomerase should be activated by genetic manipulation and fermentation control in order to elevate the HMP pathway flux, and flux towards aspartate amino acids family could be enhanced by increasing phosphoenolpyruvate carboxylase reaction rate. These may lead to a decrease in TCA flux and byproducts, and consequently the L-threonine biosynthesis would be promoted.