Urban agricultural soils are highly variable, and careful selection of sensitive indicators is needed for the assessment of soil quality. This study is proposed to develop an index based on soil enzyme activities for assessing the quality of urban agricultural soils. Top soils were collected from urban agricultural areas of Korea, and soil chemical properties, texture, microbial fatty acids, and enzyme activities were determined. The soils belonged to five textural classes with the highest frequency of sandy loam. There was no clear correlation between the soil chemical properties and soil microbial properties. Principal component analysis (PCA) and factor analysis were applied to microbial groups for identification of microbial community variation in soils. Two soil groups, namely group 1 (G1) and group 2 (G2), based on microbial community abundance were examined by PCA, and those were more prominent in factor analysis. The G1 soils showed higher microbial community abundance than G2 soils. The canonical discriminant analysis was applied to the enzyme activities of sandy loam soil to develop an index, and the index validation was confirmed using the unused soils and published data. The high-quality soils in published literature assigned the high valued index. Microbial fatty acids and soil enzyme activities can be suitable indicators for soil quality evaluation of urban agricultural soils.