[Background] Citric acid synthase is the central enzyme of carbon metabolism. It is important in the tricarboxylic acid (TCA) cycle, amino acid synthesis and glyoxylate cycle, and is a key enzyme in the synthesis of citric acid. In this paper, a citric acid-producing Aspergillus niger strain CGMCC10142 was selected. [Objective] Cloning of key genecitrate synthase, constructing the knockout strain of citrate synthase and identifying its function and influence in the process of high yield of citric acid by Aspergillus niger strain. [Methods] In this experiment, the Agrobacterium tumefaciens transformation method and the principle of homologous recombination were used, and the correct knockout strain was obtained by the dual screening method of resistance screening and lethal type and reverse screening. Observation of growth of different carbon sources by transformants and the changes of mycelial spheres and acid production during citric acid fermentation were analyzed. Finally, the effect of citrate synthase gene on the accumulation of citric acid in Aspergillus niger and its effect on the expression of important enzyme-related genes and other expressions in the main metabolic pathways were analyzed by real-time PCR. [Results] A strain T1-2 with a genetically stable knockout citrate synthase was constructed based on the high-yield citric acid strain Aspergillus niger CGMCC10142. The results showed that the strain grew slowly on the medium with glucose as carbon source and produced fewer spores. The results of shaking flask fermentation showed that the acid yield of knockout bacteria was 64.3 g/L at 84 h, which was 34.85% lower than that of the original bacteria at 98.7 g/L. Real-time quantitative PCR results showed that the expression level of citrate synthase was decreased, and the expression levels of other important enzymes were decreased. [Conclusion] The citrate synthase gene of this strain plays an important role in the accumulation of citric acid, but there are other isozyme genes. When this gene knockout, only the acid production synthesis is reduced by 34.85%. At the same time, it was found that the smooth expression of the citrate synthase facilitated the high-efficiency expression of each key enzyme in the main metabolic pathway, which laid a foundation for studying the mechanism of high-yield citric acid in Aspergillus niger.