The invention discloses a high-throughput method for segregating a quantitative character regulatory gene, which comprises the following steps of: 1) construction of a target character segregation population, in which the population is a population in two parent hybridization progenies (F2 and F3), a DH system and a RIL; 2) mixing of extreme samples and segregation of total RNA in the population, in which a progeny segregation population is divided into three categories according to character phenotype; 3) gene expression analysis, in which the difference and sameness of gene expressions between two extreme mixed samples are compared by utilizing a gene expression analysis method, namely one of chip, EST sequencing, subtraction, cDNA-AFLP, and the like; and 4) acquisition and verification of a candidate gene, in which a differential expression gene between the two extreme mixed samples is found and is a candidate regulatory gene related to target character, and the function of the gene is verified through transgene, gene expression, molecular marker correlation and a contribution rate analysis method to obtain a target gene with regulatory character phenotype. The method is suitable for the segregation of a certain quantitative character regulatory gene controlled by multigene of all organisms, and is a simple, quick, high-throughput and economical gene segregation method.