Light microscopy, immunohistochemical stain (IHC), and next-generation sequencing (NGS) were used to analyze the clinical, pathological, and molecular characteristics of a case of S100, CD34 positive, SOX10 negative soft tissue sarcoma with EML4-ALK fusion. The patient is a 68-year-old man with an intact capsular mass in the subcutaneous adipose tissue of the right thigh. Histologically, the tumor cells were short spindle and oval with vortex-like, small nested, or scattered distribution, and had myxoid, spindle fibrous stroma, or homogeneous hyalinosis stroma. The mitosis were active, showing pathological mitosis. IHC showed CD34, ALK, CD99, H3K27me3 diffusely positive, S100 focal positive, SOX10, STAT6, CD31, SMA, Desmin negative, Ki-67 proliferation index was about 30%. NGS detected 2 gene fusions, EML4-ALK and EIF4E3-FOXP1. Combined with literature review, malignant peripheral nerve sheath tumor, dermatofibrosarcoma protuberans, and solitary fibrous tumor could be excluded, and S100, CD34 positive soft tissue sarcoma with EML4-ALK fusion was diagnosed. Spindle cell tumors characterized by S100, CD34 positive and SOX10 negative are a group of tumors with different gene fusions, different histological morphology, and different proliferation states. Their biological behaviors are diverse. Molecular detection is essential for their diagnosis and differential diagnosis. It can also provide a basis for targeted therapy.