Exosomes are a type of extracellular vesicles containing mRNA, miRNA, and proteins of origin cells, which can control the characteristics of other cells or surroundings. Despite increasing evidence on oncogenic properties of tumor-derived exosomes, fibrosarcoma-derived exosomes remain largely unrevealed. While the proper extraction and characterization of exosomes is critical in exosomes research, there are various limitations in techniques to measure the size and homogeneity of exosomes. Here, we analyzed exosomes from a fibrosarcoma cell line WEHI-164 compared with a breast cancer cell line MDA-MD-231 as a control. Results from dot blot and western blot analysis demonstrated that GM1 ganglioside, and TSG101, HSC70 and GAPDH proteins were contained in exosomes from the WEHI-164 fibrosarcoma cell line. The existence of tetraspanins such as CD81, CD63 and CD9 was confirmed in the exosomes by ExoView analysis. The results obtained from TEM showed their sphere-like shapes of around 50 to 70 nm in radius. Through DLS, we found out that the mean radius of the exosomes derived from WEHI-164 and MDA-MB-231 cell lines was 94.4 nm and 107.8 nm, respectively, with high homogeneity. When comparing the radius measured by TEM with the radius measured by DLS, it was revealed that the difference between the two methods was about 40 nm. This study has significance in characterizing the molecular properties of exosomes from a fibrosarcoma, which has not been researched much before, and in providing clear evidence that DLS can be used as an efficient, convenient and noninvasive technique to simply check the homogeneity and size of exosomes.