Aquaculture discharge is an important non-point source for antibiotic contamination, yet antibiotic removal from aquaculture wastewater has not been given the necessary attention. Here, nine constructed wetlands (CWs) with different configurations (i.e., substrate, plants, and hydraulic retention time) were prepared by orthogonal test [L9 (33)], to treat aquaculture wastewater and specifically to evaluate the removal efficiency and mechanism of antibiotics. Consequently, the highest removal percentages of trimethoprim (89 ± 3%), sulfamethoxazole (61 ± 7%), sulfamonomethoxine (20 ± 8%), sulfamethazine (20 ± 9%), and sulfadiazine (12 ± 13%) were achieved via collocating optimal configuration of CWs. Based on the analysis of both the orthogonal test and correlation, microbial degradation was the major removal pathway of antibiotics, particularly anaerobic processes. Further, 16S rRNA sequencing analysis presented that some specific substrate, plants, and hydraulic retention time might promote antibiotic removal via changing dominant and functional microbes by deciding oxygen concentrations and redox conditions in CWs. In which, anaerobic bacteria (e.g., Lacihabitans and Ilumatobacter) were regarded as the key microorganisms in the genus level for removing antibiotics by CWs through redundancy analysis (RDA), while aerobic bacteria (e.g., Hydrogenophaga and Pseudomonas) were likely to significantly inhibit the anaerobic process of antibiotic degradation.