Manure application increased the abundances of ARGs and MGEs in agricultural soils. Five classes of ARGs and two MGEs were prevalent in manured and unfertilized soils. Genera Pseudomonas and Bacteroidetes might be the dominant hosts of intI1 and ermF. The abundances of ARGs positively correlated with TC, TN, OM, Cu, Zn, Pb and MGEs. Land application of manure tends to result in the dissemination of antibiotic resistance in the environment. In this study, the influence of long-term manure application on the enrichment of antibiotic resistance genes (ARGs) and mobile genetic elements (MGEs) in agricultural soils was investigated. All the analyzed eight ARGs (tetA, tetW, tetX, sulI, sulII, ermF, aac(6’)-Ib-cr and blaTEM) and two MGEs (intI1 and Tn916/1545) were detected in both the manured and control soils, with relative abundances ranging from 10-6 to 10-2. Compared with the control soil, the relative abundances of ARGs and MGEs in manured soils were enriched 1.0–18.1 fold and 0.6–69.1 fold, respectively. High-throughput sequencing analysis suggested that at the phylum level, the bacteria carrying intI1 and ermF might be mainly affiliated with Proteobacteria and Bacteroides, respectively. The dominant genera carrying intI1 and ermF could be Pseudomonas and Bacteroides, independent of manure application. Correlation analysis revealed that ARGs had strong links with soil physicochemical properties (TC, TN, and OM), heavy metals (Cu, Zn and Pb) and MGEs, indicating that the profile and spread of ARGs might be driven by the combined impacts of multiple factors. In contrast, soil pH and C/N exhibited no significant relationships with ARGs. Our findings provide evidence that long-term manure application could enhance the prevalence and stimulate the propagation of antibiotic resistance in agricultural soils.