The present study aims to evaluate the potential of transcriptomic profiles in evaluating the impacts of complex mixtures of pollutants at environmentally relevant concentrations on aquatic vertebrates. The changes in gene expression were determined using microarray in the liver of male zebrafish (Danio rerio) exposed to surface water collected from selected locations on the Hun River, China. The numbers of differentially expressed genes (DEGs) in each treatment ranged from 728 to 3292, which were positively correlated with chemical oxygen demand (COD). Predominant transcriptomic responses included peroxisome proliferator-activated receptors (PPAR) signaling and steroid biosynthesis. Key pathways in immune system were also affected. Notably, two human diseases related pathways, insulin resistance and Salmonella infection were enriched. Clustering analysis and principle component analysis with DEGs differentiated the upstream and downstream site of Shenyang City, and the mainstream and the tributary sites near the junction. Comparison the gene expression profiles of zebrafish exposed to river surface water with those to individual chemicals found higher similarity of the river water with estradiol than several other organic pollutants and metals. Results suggested that the transcriptomic profiles of zebrafish is promising in differentiating surface water with pollution gradient and different discharges and in providing valuable information to support discharge management.