The invention relates to a multiple-gene helicobacter pylori detection system and a kit and application thereof. The helicobacter pylori detection system includes 21 pairs of primers respectively for a strain identification gene (16S rRNA), virulence genes (cagA, vacA-s1, vacA-s2, vacA-m1, vacA-m2, iceA1, iceA2, dupA, oipA and luxS), drug resistance genes (2143 locus of 23S rRNA, 148 locus of rdxA, 1777 locus of pbp1A and polymorphism of the 261 locus of gyrA) and quantitative analysis genes ureC and beta-globin of the helicobacter pylori. The multiple-gene helicobacter pylori detection system and the kit of the system do not need the steps including conventional isolated culture and the like, synchronous detection and analysis on strain identification, quantification, virulence and medicine resistance can be directly conducted on tissue samples in the same reaction system, the shortcomings of low flux, long consumed time, low detection rate and the like of a conventional detection method are overcome, a comprehensive, accurate and low-cost etiological diagnosis is clinically provided for the first time, and important references are provided for individualized diagnosis and accurate treatment of helicobacter pylori infection.