To investigate the effect of long-term manure application on the profiles of antibiotic resistance genes (ARGs) in paddy soil, the abundance of ARGs targeted by 295 primers sets were measured by using high-throughput quantitative PCR technique. A total of 107 types of ARGs were detected in long-term manured paddy soil, which is significantly higher than that in control paddy soil (P < 0.05). PCA analysis and UPGMA cluster demonstrated that long-term manure application significantly changed the ARGs diversity and abundance in paddy soils. There were 49 types antibiotic resistance genes were enriched significantly in paddy soil applied with manure (P < 0.05), especially the mexF, which belongs to quinolones-chloramphenicol resistance genes, increased 1791-fold compared with paddy soil of no manure application. Our results showed that long-term application of manure in paddy soil resulted in the shift of abundance and diversity of ARGs, high-throughput quantitative PCR could be a powerful tool concerning the environmental ARGs dimensions.