Global spread of antibiotic resistance has hampered the infection therapies by antibiotics and posed great threat to human health. Agroecosystem is the source and sink of antibiotic resistance genes (ARGs) and strongly associated with human health. Antibiotics have been extensively used in human medicine and animal production for prevention and treatment of disease, and promotion of animal growth. Animal farms and sewage treatment plants are the hotspots for dissemination of ARGs in environments. Application of animal wastes and sludges contributes largely to the increased resistance in agroecosystems via directly introduction of antibiotic resistant bacteria or ARGs, or through introduction of antibiotic residues and selection of indigenous resistant bacteria. However, discriminating their contribution to the ARGs level in agroecosystem is difficult. Both culture-dependent and culture-independent technologies, including metagenomic, microarray and high-throughput quantitative polymerase chain reaction have been utilized in the characterization of ARGs in agroecosystem, and each has its strength and weakness. Numerous researches have investigated the diversity and abundance of ARGs in agroecosystems. However, the baseline or background data of ARGs and the knowledge of biogeography of ARGs in agroecosystems are limited. Lack of standard surveillance system hinders the comparison of results from various studies. Horizontal gene transfer (HGT) plays a pivotal role in the dissemination of ARGs in environments. Plasmids, integrons, bacterial phages and extracellular DNA are important mobile genetic elements (MGEs) mediating HGT in agroecosystems. The concentration of antibiotics, heavy metals and other chemicals contribute to the HGT of ARGs. The primary risk of ARGs is that they could be transferred from environmental bacteria to human or animal pathogens, resulting in antibiotic treatment failure. However, knowledge gaps remain in evaluating the impact of ARGs in agroecosystems to human health. ARGs in environments are ubiquitous and diverse, prioritizing the risk of ARGs in the environments is an essential step to human health risk assessment (HHRA). Then to investigate the dissemination of these highly risk ARGs, particularly estimate the likelihood of their introduction into human pathogens is necessary. Finally, the lack of quantitative estimates of human exposure to these ARGs and their link with large-scale epidemiological survey are the key data gaps in the assessment of human health risk.