To reveal the temporal and spatial variation pattern of groundwater chemistry in Lihu Lake and explore the causes for the change of water quality through analysis of sulfur isotope and nitrogen-oxygen isotope, so as to provide scientific basis for reasonable exploitation and protection of karst water resources. Several groundwater samples, collected from January to December in 2010 and May, October in 2014 were monitored to analyze the chemical composition of conventional water and the characteristics of delta34S-SO4(2-), and delta15N-NO3-. The results showed that: (1) The hydrochemical type of the underground water was HCO3-Ca type and effected by the seasonal precipitation and human activity, the temporal and spatial variation of the main cations was obvious. (2) The sulfur concentration in the underground river was slightly decreased since the operation of the sewage plant, however, the sulfur concentration was still high. The nitrate pollution aggravated in the year 2014 compared to 2010. Impacted by human activity, the concentration of sulfur and nitrate was higher in the upstream Nandan river, Layi cave and the midstream Liangfeng cave, and Gantianba than in the downstream Xiaolong cave. (3) The delta34S-SO4(2-) value ranged from -4.12%o to -0.93%o. It was inferred that the emission of sulfur oxides through burning coal resulted in the rainwater acidification, which input a large amount of SO4(2-) into the underground water in the form of acid rain. (4) The delta15N-N03- value ranged from 0.26%o to 11.58%o, with an average value of 7.61%o, the delta18O-NO3- value ranged from -2.33%o to 21.76%o, with an average value of 9.38%o. In combination of the composition analysis of nitrogen-oxygen isotope of nitrate, it was believed that soil organic nitrogen, manure and sewage were the main sources of nitrate in the groundwater and the main causes for aggravation of nitrate pollution of the underground water.