Accurate and comprehensive estimation of agricultural non-point source pollution (ANPSP) is of vital importance in watershed pollution control, while data inaccessibility and complexity of process-based models may be main restrictions in practice. An estimation methodology framework for ANPSP was established based on improved export coefficient model (IECM) in this research. A series of updated, regional coefficients were collected and/or calculated with multiple pollution sources considered (i.e., animal husbandry, farming and forestry, fishery, and rural domestic sewage) to provide a comprehensive estimation of TN and TP pollution loads in Taihu Lake Basin. According to this model, pollution loads of TN and TP in Taihu Lake Basin were 65791.28 and 11400.38 t in 2016, respectively. Animal husbandry was main source of ANPSP, accounting for 31.05% and 66.70% for TN and TP, respectively, which requires more efficient and clean agricultural production modes and strict pollution control measures. IECM-based estimation framework may provide a reference for ANPSP management practice in large scale watershed.