The invention discloses a metagenome 16S hypervariable region V3 based classification method and a device thereof. The method contains the following steps of: extracting DNA in microbial samples; carrying out amplification of metagenome 16S rDNA hypervariable region V3, carrying out Solexa database construction on amplification products, and simultaneously marking each sample by adding a connector with a label sequence in the process of database construction; mixing different samples with label sequences, sequencing by the use of a Solexa sequencing tool after mixing to obtain original sequencing sequences reads distinguished by labels; assembling by the use of reads overlapping relations to obtain hypervariable region V3 full-length sequences unique reads; and carrying out classification analysis on unique reads to accomplish classification of microbial population. By the adoption of the method and the device provided by the invention, classification of microbial population is accurate and sequencing cost is greatly reduced.