Fecal-contaminated water may enter the food chain and become an important route for the transmission of antibiotic resistance genes (ARGs) to the human microbiome. However, little is known about the spread of ARGs from fecal contamination in water bodies along the aquatic food chain. In this study, laboratory-raised Daphnia magna and Aristichthys nobilis were used to investigate the effects of the addition of manure on target ARGs in water and their intestinal contents to determine the potential transmission route of ARGs in the aquatic food chain system. The abundance of target ARGs in water as well as D. magna and A. nobilis intestinal contents significantly increased when fecal contamination was present. ARGs bioaccumulated along the food chain, with four ARGs (tetM-01, tetX, qnrS, and sul2) detected regularly. Mn and Cr were key environmental factors that promoted the transfer of ARGs along the food chain. Fecal addition significantly changed the structure of microbial communities in water, D. magna gut, and A. nobilis gut. The ARG spectrum was significantly correlated with the composition and structure of the bacterial community. Proteobacteria, Bacteroidetes, and Firmicutes were identified as the main host bacteria and were likely to act as carriers of ARGs to promote the spread of antibiotic resistance in the food chain. The composition and structure of bacterial communities, along with mobile genetic elements, were two key drivers of ARG transfer. These findings provide new insights into the distribution and spread of ARGs along the freshwater food chain.