Agricultural runoff is an important antibiotic resistance genes (ARGs) dissemination pathway from farmlands to water environment, however few studies have focused on the influence of agricultural land-use change on the pattern of ARGs in runoff and assess the health risk to public. Lake Tai Basin which experiences agricultural land-use change was selected to elucidate this concern. Our findings revealed that the pattern of ARGs was more diverse and the gene abundance was higher in orchard runoffs by comparison with conventional cropland runoffs. Co-occurrence network analysis between mobile genetic elements and ARGs demonstrated that after agricultural land-use change, ARG dissemination via runoffs became more threatened. In addition, this study illustrated the correlations between the antibiotic resistome and microbiome in runoffs, finding that non-dominant microbial taxa were the limiting factor which determined the pattern of ARGs in surface runoffs. In summary, the pattern and dissemination risk of ARGs in the surface runoff after agricultural land-use change in Lake Tai Basin were clarified via this study. Display omitted •Orchard runoffs harbor more diverse and abundant ARGs than traditional cropland runoffs.•After land-use change, runoffs possess a strong ability for disseminating ARGs in Lake Tai Basin.•Non-dominant bacterial taxa are closely correlated with the antibiotic resistome in runoffs.•The effect of agricultural land-use change on the ARG contamination in Lake Tai Basin should not be underestimated.