Based on the community structure of macrozoobenthos in the Liuxi River, which was surveyed during the pre-flood, latter flood, and dry seasons in 2016, the Benthic-Index of Biotic Integrity (B-IBI) was first used to evaluate the ecological health of this subtropical river in China. Overall, 103 species of zoobenthos were identified, belonging to four phyla, eight classes, 22 orders, 52 families, and 94 genera. Selected from 32 candidate metrics, the B-IBI system of the Liuxi River was constructed using five key indicators, including total biomass, individual percentage of sensitive groups, and taxa number for EPT, Chironomidae, and tolerant groups. The health criteria were > 3.24, healthy; 3.24-2.43, sub-healthy; 2.43-1.62, fair; 1.62-0.81, fair-poor; < 0.81, poor. The proportion of healthy sites was 14.29%, sub-healthy sites 50.0%, good-fair sites 21.42%, fair sites 14.29%, and there were no poor sites. In general, the assessment of the B-IBI for the Liuxi River suggested that 1) the environments in the headwaters and upper stream had healthy conditions, with EPT taxa and sensitive groups contributing the most to B-IBI scores, and 2) the environments in the lower reaches deteriorated seriously, with the taxa number of tolerance groups contributing most to the B-IBI scores. The correlation analysis showed that the B-IBI values were positively correlated with dissolved oxygen (P < 0.01), but negatively correlated with electric conductivity, ammonia, total nitrogen, and phosphorous (P < 0.05). Canonical correspondence analysis showed that the first two axes of the B-IBI indicators explained 68.1% of environmental variables, which clearly distinguished the upstream from downstream, as well as main stems and tributaries in terms of health. Our results suggested that the B-IBI system constructed in this paper could objectively and precisely reflect the ecological health and water quality of the Liuxi River. In addition, compared with the subtropical climate zones where the Liuxi River is located, the influences of urban development and human disturbances on the B-IBI were much more serious, indicating the applicability of the B-IBI in different geographical regions.