Antibiotics are extensively utilized in aquaculture to mitigate diseases and augment the productivity of aquatic commodities. However, to date, there have been no reports on the presence and associated risks of antibiotics in the emergent rice-crayfish rotation (RCR) system. This study investigated the occurrence, temporal dynamics, prioritization, sources, and potential for resistance development of 15 antibiotics within the RCR ecosystem. The findings revealed that during the crayfish breeding and rice planting periods, florfenicol (FFC) predominated in the RCR's surface water, with peak and average concentrations of 1219.70 ng/L and 57.43 ng/L, and 1280.70 ng/L and 52.60 ng/L, respectively. Meanwhile, enrofloxacin (ENX) was the primary antibiotic detected in RCR soil and its maximum and average concentrations were 624.73 ng/L and 69.02 ng/L in the crayfish breeding period, and 871.27 ng/L and 45.89 ng/L in the rice planting period. Throughout the adjustment period, antibiotic concentrations remained relatively stable in both phases. Notably, antibiotic levels in surface water and soil escalated during the crayfish breeding period and subsided during the rice planting period, with these fluctuations predominantly influenced by FFC and ENX. Source analysis indicated that the antibiotics in RCR predominantly originated from aquaculture activities, supplemented by water exchange processes. Utilizing the entropy utility function and a resistance development model, FFC, clarithromycin (CLR), and roxithromycin (ROX) in surface water, along with ENX, CLR, and ROX in soil, were identified as priority antibiotics. FFC, ENX, and ROX exhibited a medium risk for resistance development. Consequently, this study underscores the necessity to intensify antibiotic usage control during the crayfish breeding period in the RCR system to mitigate environmental risks.