Extracellular vesicles (EVs) from antler reserve mesenchymal (RM) cells play an important role in the paracrine regulation during rapid growth of antler without forming a tumor; therefore, RM-EVs become novel materials for anti-tumor studies, such as osteosarcoma treatment. However, the problem of low production of RM-EVs in traditional 2D culture limits its mechanism research and application. In this study, we established an optimal 3D culture system for antler RM cells to produce EVs (3D-RM-EVs). Morphology and property of harvested 3D-RM-EVs were normal compared with EVs from conventional 2D culture, and the miRNA profile in them was basically the same through transcriptome sequencing analysis. Based on the same number of RM cells, the volume of the culture medium collected by 3D cultural system concentrated nearly 30 times, making it more convenient for subsequent purification. In addition, EVs were harvested 30 times in 3D cultural system, greatly increasing the total amount of EVs (harvested a total of 2-3 times in 2D culture). Although 3D-RM-EVs had a limited inhibitory effect on the proliferation of K7M2 cells, the inhibition effect of 3D-RM-EVs loaded drugs (Ifosfamide + Etoposide) were more significant than that of positive drug group alone ( < 0.05). Furthermore, studies showed that 3D-RM-EVs loaded drugs (Ifosfamide + Etoposide) had the most significant tumor inhibition effect, with decreased tumor size, and could slow down body weight loss compared with Ifosfamide + Etoposide (IFO + ET) group. These results demonstrated that 3D-RM-EVs were efficiently prepared from antler RM cells and were effective as drug vehicles for the treatment of osteosarcoma.