The occurrence and spread of antibiotic resistance genes (ARGs) are concerns that have threatened public health for many years. However, the effects of humic acid (HA) application on the expression of ARGs in chlorophenols wastewater treatment are rarely reported. In this study, we investigated the sludge performance, including the removal of pollutants, changes in the microbial communities, and the expression of tetracycline resistance genes (TRGs), to explore the function of HA in 4-chlorophenol (4-CP) wastewater treatment at different HA concentrations. The results showed that HA application did not significantly stimulate the removal of pollutants, other than the removal of PO-P. High-throughput sequencing analysis indicated that the application of HA influenced the microbial communities and changed the expression level of TRGs. Quantitative real-time PCR analysis showed that the expression of numerous TRGs (tetC, tetG, tetW, tetX, and intI1) was significantly inhibited by the application of HA (25 mg L) during 4-CP wastewater treatment. In summary, HA application played an important role in treating chlorophenols wastewater and reducing the expression of TRGs. This work aimed to provide an efficient method of reducing the expression level of ARGs in industrial wastewater treatment, which has inevitable environmental significance.