The invention relates to a method and a kit for enriching 4000 human pathogenic target genes. The method comprises the following steps: breaking DNA to obtain DNA fragments, carrying out terminal repairing, carrying out A addition, carrying out linker addition, and carrying out Pre-Capture LM-PCR; mixing Pre-Capture LM-PCR products of 4000 human pathogenic target genes to obtain a mixed library sample, hybridizing a specific probe library of the 4000 human pathogenic target genes with the mixed library sample, capturing DNA by adopting magnetic beads, and carrying out eluting; and carrying out Post-Capture LM-PCR. The kit contains a DNA terminal repairing reagent, an A addition reagent, a linker addition reagent, an LM-PCR reagent and a capturing reagent. According to the method and the kit, the 4000 human pathogenic genes are taken as target spots and are combined together, the gene sequences are enriched once through a biotin probe capturing technology and are subjected to high-throughput sequencing, therefore, multiple variation types of the genes can be simultaneously examined, and the detection sensitivity is high; the 4000 pathogenic genes can be taken as a principal diagnosis and detection means clinically, the cost is reduced, the missed diagnosis is reduced, the detection positive rate is improved, and therefore, the clinical popularization is easy.