The invention discloses a high-throughput targeted library building method and application. The high-throughput targeted library building method comprises the following steps that a targeted capture probe and a PCR amplification primer are designed and synthesized based on a detected gene; a genome DNA of a sample is hybridized with the targeted capture probe overnight to obtain a captured targetfragment, and cyclizing, digesting, purifying and PCR amplification are carried out to obtain a constructed targeted sequencing library; and the targeted sequencing library is purified, the concentration and size of DNA fragments of the library are detected, second-generation sequencing is carried out, and the quality and sequencing information of the targeted sequencing library are analyzed according to a sequencing result. According to the high-throughput targeted library building method and application, hundreds of detection genes can be amplified in a round of PCR reaction, so that the defects that only a small number of gene loci can be detected by multiplex PCR library construction and experimental conditions are difficult to optimize are overcome; only one turn of PCR is performed,and random barcode are arranged on a probe framework, so that PCR bias is reduced, and base errors introduced by PCR and sequencing can be distinguished; and the library building step is simplified, and the reagent and time cost is reduced.