Nutrient enrichment from nonpoint source pollution is one of the main causes of poor water quality and biotic impairment in many streams and rivers worldwide. The establishment of reference nutrient conditions in a river system is an essential but difficult task for water quality control. In the present study, the reference concentrations of total nitrogen (TN) and total phosphorus (TP) were estimated in an intensive agricultural watershed, the Cao-E River system of Eastern China. Based on a 3-year water quality monitoring data in the river system, three approaches were adopted to establish the reference concentrations of TN and TP, those are the 75th percentile of frequency distribution of nutrient concentrations in reference streams, the 25th percentile of frequency distribution of nutrient concentration in general streams (including reference and non-reference streams) and regression modeling. Results showed that the nutrient reference concentrations were slightly different from different approaches. By the three approaches, the average reference concentrations for TN and TP in the study system were 1.73 ± 0.13 mg l(-1) and 55.23 ± 4.77 μg l(-1) with CV of 7.39 % and 8.63 %, respectively. Accordingly, the reference concentrations for TN and TP were recommended to be 1.70 mg l(-1) and 55 μg l(-1), respectively. In the mountainous and intensive agricultural watershed, the major anthropogenic impacts to river water quality were the urban area percentage cover, cropland area with slopes 0-8°, and livestock and poultry waste loads density. These variables could account for 89.7 % and 80.3 % of the total variations for TN and TP concentration, respectively.