The invention discloses a DNA library for detecting MO pathogenic gene mutations through a targeted high-throughput semiconductor sequencing technique and application of the DNA library. Specifically, a primer pool is designed according to 15 MO pathogenic genes, sample genome DNA is subjected to super-multiplex PCR amplification, and an amplified product is sequenced by using the high-throughput semiconductor sequencing technique to find pathogenic mutations so as to provide a theoretical basis of genetics and molecular biology for clinical diagnosis. The DNA library for detecting the MO pathogenic genes disclosed by the invention has the characteristics of accuracy, rapidness, flexibility and low cost, and 15 gene detection regions related in the invention comprehensively cover all known pathogenic genes of MO, thereby having important significance and clinical values in diagnosis and differential diagnosis of MO.