Soil fauna play a crucial role in sustaining agro-ecosystem functions. Crop straw is recommended for application to agricultural fields to improve soil quality. However, the effects of crop straw combined with different synthetic fertilizers on the soil faunal community remain unclear, and knowledge regarding purple soil is limited. Using the conserved cytochrome c oxidase I (COI) gene as markers, we examined the responses of the soil faunal community to different fertilization in upland purple soil of southwestern China. The accuracy of the morphological and molecular methods in characterizing soil nematodes was compared. Our results showed that different fertilization treatments significantly changed the soil faunal community structure (Adonis test, R-2 = 0.43, P = 0.011). Sixteen biomarkers were identified according to LEfSe (linear discriminant analysis effect size). The diversity and species number of soil fauna were closely related to soil organic matter (SOM) and total phosphorus (TP) (P < 0.05). This study indicates that crop straw return can improve the soil fertility and diversity of soil fauna in purple soil. Additionally, the morphological approach and molecular method based on the COI gene can be considered as complementary approaches in characterizing soil nematode community.