In the past decade, with the rise of research on plant-derived extracellular vesicles (PDEVs), scientists have been continuously exploring the bioactivity of PDEVs. Many PDEVs have been shown to possess a variety of biological activities. Given that the specific characteristics of EVs are believed to be related to their source cells, PDEVs from traditional Chinese medicinal herbs hold significant potential for development. In this study, lotus ( Gaertn.) leaves were selected as the source of PDEVs, and the impact of different isolation methods on their characteristics was evaluated, while their potential biological activities were also assessed. Lotus-derived EVs (LDEVs) were isolated by using tangential flow filtration (TFF), ultracentrifugation (UC), density gradient ultracentrifugation (DGU), and size-exclusion chromatography (SEC), respectively. The mean sizes of LDEVs isolated by various methods were in the range of 130-160 nm. Although the LDEVs isolated by the TFF method had a lower zeta potential, it exhibited the highest purity, with a yield of 3.69 ± 0.43 × 10 particles/g lotus leaves. Notably, LDEVs isolated by different methods all demonstrated the ability to attenuate LPS-induced inflammation in RAW264.7 cells, significantly decreasing the nitrite concentration in the culture medium. Furthermore, LDEVs also showed potential for wound healing, promoting the migration of HaCaT cells in vitro. LDEVs also demonstrated internalization by RAW264.7 and HaCaT cells. These results support the potential of LDEVs for biomedical applications while also suggesting that TFF is a promising and viable strategy for large-scale PDEV isolation.