The invention discloses a method for determining absolute abundance of a microbial community structure in an environment. The method comprises the following steps: extracting DNA in an environmental sample, obtaining a DNA sample, and determining a total concentration of classification genes in the DNA sample through a real-time fluorescent quantitative PCR (Polymerase Chain Reaction) method; obtaining the relative abundance of the classification genes in microbes of different kingdoms, phyla, classes, orders, families, categories and species through a second-generation high-throughput sequencing method; and finally, multiplying the total concentration of the classification genes in the DNA sample by the relative abundance of the classification genes in the microbes of different kingdoms, phyla, classes, orders, families, categories and species, and calculating to obtain the absolute abundance of the classification genes in the microbes of different kingdoms, phyla, classes, orders, families, categories and species, so that rapid high-throughput determination of the absolute abundance of the microbial community structure is realized. Therefore, cross-group sample comparison is reliable, growth and decline of the species can be substantially reflected, and a technical support is provided for further exploration of the microbial community structure.