Abstract: The plants and their root microorganisms in the constructed wetland have a good removal effect on the antibiotics in the environment, but the understanding of the response characteristics of various plants and microorganisms under antibiotic stress is still insufficient and unclear, so it is necessary to clearly study the differential response characteristics of plants and microorganisms under different concentrations of antibiotic stress. The removal performance of ofloxacin (OFL) by five wetland plants (Gladiolus hybrids, Cyperus alternifolius, Scirpus validus Vahl, Oenanthe javanica, Juncus effusus L.) microbial systems under the stress of OFL, a typical polar antibiotic, was studied. Based on the removal efficiency of OFL by different plants, the root activity, active oxygen and antioxidant system of Gladiolus hybrids and Cyperus alternifolius were studied, and the response characteristics of their root microorganisms under OFL stress were analyzed. The results showed that the five wetland plants had significant differences in the removal of OFL, among which the Cyperus alternifolius had the best removal performance of OFL, followed by Gladiolus hybrids. The plant-microbial systems showed different sensitivities under OFL stress of different concentrations. Taking 10 mg/L as the turning point, when OFL concentration was below 10 mg/L, it could promote plant root growth, plant root activity and root antioxidant enzyme activity. When OFL concentration exceeded 10 mg/L, Gladiolus hybrids and Cyperus alternifolius roots were poisoned by OFL, and plant root activity and antioxidant enzyme activity were inhibited. The measurement results of root microbial community using high-throughput sequencing technology showed that low concentration OFL (1 mg/L) was positively correlated with plant root microbial community diversity and species richness, while high concentration OFL (50 mg/L) was negatively correlated. Using PICRUSt function software to predict the function of microbial community, it was found that high concentration OFL stress could accelerate the metabolic rate of plant root microorganisms.