Agricultural non-point source pollution refers that substance such as nitrogen and phosphorus cause water environment pollution through surface runoff and underground leakage in agricultural production activities. Water environment pollution related to agricultural non-point source pollution in the Huaihe River Basin is becoming more and more prominent. Therefore, it is necessary to analyze the characteristic of soil nutrient in cultivated land and explore the spatial variation and influencing factors of soil nutrients at the watershed scale. A total of 239 topsoil samples were collected from the Guo river basin, and the related factors of soil organic matter (SOM), total carbon (TC), total nitrogen (TN), total phosphorous (TP), total potassium (TK) and potential of hydrogen (PH) were studied by using descriptive statistics and geostatistical methods. The results showed that TK and PH were weak variation, while SOM, TC, TN and TP were medium variation. Soil pH, TP, TK, TC and SOM had moderate spatial variability, which was caused by both random factors and structural factors such as soil texture, soil type, fertilization and local ecological restoration management. Soil TN showed a strong spatial correlation, mainly due to soil texture and soil type. If the recommended fertilization amount is still given based on the average value of soil nutrients ignoring the spatial heterogeneity, it will not only affect crop production efficiency and fertilizer utilization, but may also cause greater environmental pollution. This study can provide a theoretical basis for the management of agro-ecological environments throughout the basin area.