Tracing water sources of streamflow in a mixed land-use catchment is critical for predicting pollutant emissions from various human activities to streams but remains a major challenge. A rain event based field monitoring study was conducted in the Jieliu catchment located in the hilly area of central Sichuan Province, southwest China. The ratio of the maximum fluorescence intensities (Fmax) of the two humic-like dissolved organic matter (DOM) components at excitation/emission wavelengths of 255 (315)/415 nm (component 1; C1) and 260 (375)/ 480 nm (component 2; C2) was proposed as a tracer for quantifying streamflow water sources. Satisfactory performance of using the Fmax(C1)/Fmax(C2) ratio in hydrograph separation of streamflow at the outlet of a forest sub-catchment was verified by through comparison with the hydrograph separation results based on delta 18O data. The Fmax(C1)/Fmax(C2) ratio was then applied to estimate the contributions of rainwater and pre-event water sources under different land use types to the streamflow in an agro-forest sub-catchment and the entire catchment. The hydrograph separation results using the Fmax(C1)/Fmax(C2) ratio can be used to support the optimization of water resource management and the quantification of pollutant loadings from major water sources to streams at the catchment scale.