Metabolic engineering of various industrial microorganisms to produce chemicals, fuels, and drugs has raised interest since it is environmentally friendly, sustainable, and independent of nonrenewable resources. However, microbial metabolism is so complex that only a few metabolic engineering efforts have been able to achieve a satisfactory yield, titer or productivity of the target chemicals for industrial commercialization. In order to overcome this challenge, ¹³C Metabolic Flux Analysis (¹³C-MFA) has been continuously developed and widely applied to rigorously investigate cell metabolism and quantify the carbon flux distribution in central metabolic pathways. In the past decade, many ¹³C-MFA studies have been performed in academic labs and biotechnology industries to pinpoint key issues related to microbe-based chemical production. Insightful information about the metabolic rewiring has been provided to guide the development of the appropriate metabolic engineering strategies for improving the biochemical production. In this review, we will introduce the basics of ¹³C-MFA and illustrate how ¹³C-MFA has been applied via integration with metabolic engineering to identify and tackle the rate-limiting steps in biochemical production for various host microorganisms