Abstract: The “Three line” of land spatial planning and the spatial characteristics of cultivated land quality can greatly contribute to the spatial layout optimization of cultivated land protection and food security. In this study, an improved local spatial autocorrelation model was proposed to optimize the spatial layout of arable land protection using “Three line” delineation of territorial spatial planning and spatial autocorrelation attributes of arable land quality. The ecological environment served as the “fourth dimension” of the spatial correlation analysis of cultivated land quality. A plan was also presented to improve the spatial layout of cultivated land protection. The spatial autocorrelation correlation was then simulated for the natural quality, utilization management, economic value, and ecological environment index of cultivated land in the “Three line” from the plot scale. There was a positive influence on the geographical evolution of agricultural ecological landscape patterns, food safety, and farmland pollution control. The specific procedures were as follows. Firstly, some indexes were estimated to obtain the three-line delineation of land spatial planning, including the natural quality, utilization management, economic value, and ecological environment index. The plot data was collected from 1 073 soil monitoring stations in the Gaochun District, Nanjing City, Jiangsu Province of China. Secondly, the spatial correlation of each indicator was analyzed using the spatial error model of the enhanced spatial weights. Finally, a new strategy was proposed to optimize the spatial layout of cultivated land, according to the geographical association findings of permanent basic farmland, urban development boundary, and the quality of inland blocks of ecological protection red line. The results indicated: 1) Much more high-quality cultivated land was concentrated in the west and dispersed in the east, in terms of the geographical distribution of cultivated land quality. Low-quality agricultural land was more prevalent in the eastern part than in the western. The ecological environment, economic value, utilization management, and natural quality index all demonstrated the "west high, east low" features of geographical distribution. 2) Each cultivated land quality index presented a positive geographic correlation, according to the spatial autocorrelation analysis of the cultivated land quality index. Both positive and negative correlation types were quite compatible with the spatial distribution of high and low-quality cultivated land. The natural quality, utilization management, economic value, and ecological environment index all presented the Moran's I values of 0.79, 0.92, 0.89, and 0.77, respectively, all of which were the spatial aggregation features. The indexes were ranked in descending order of the Utilization Management, Economic Value, Natural Quality, and Ecological Environment Index. 3) The cultivated land was divided into 14 second-level categories and four first-level categories using the spatial correlation of the cultivated land quality, including the permanent basic farmland protection, urban development buffer, ecological environment protection, and comprehensive adjustment zone. Both the permanent basic cropland and the grade rose by 0.94. The best quality was found in the permanent basic farmland protection zone. There was a significantly positive spatial dispersion impact of each quality measure for the cultivated land protection to forbid non-agricultural building. The urban development buffer zone was the best place for urban growth, due to the low quality of the farmed land and the significant geographical benefit. The ecological environmental protection zone was utilized to carry out ecological protection in the field. An ecological red line protection grid was constructed for the outstanding ecological circumstances, especially with a relatively visible deficit in the overall quality.