Objective To detect and analyze the plasma levels and immunoactivities of different forms of circulating-free DNA (cfDNA) in systemic lupus erythematosus (SLE) patients. Methods The study enrolled 58 patients with SLE, 66 patients with other autoimmune diseases (non-SLE) and 60 healthy individuals. Total cfDNA, exosome cfDNA and immune complex cfDNA were extracted from the plasma and detected using a fluorescence method. Overall methylation levels of cfDNA were measured. Macrophages and dendritic cells induced in vitro were co-cultured with exosomes or immune complexes derived from SLE patients pre-treated with deoxyribonuclease 1-like 3(DNASE1L3) or immunoglobulin G (IgG) specific degradation enzyme or none. Then, cytokines and cell surface activation markers were detected by the multiple fluorescent microsphere assay. Results Among the three groups, SLE patients had the highest levels of exosomes and immune complex cfDNA, followed by non-SLE patients, and no significant differences were found in the simple cfDNA. The methylation levels of different types of cfDNA (except simple cfDNA) in the plasma of SLE patients were significantly lower than that of the non-SLE group and healthy individuals. Non-SLE patients and healthy individuals presented no significant differences for the methylation levels of all cfDNA types. After DNASE1L3 and IgG enzyme treatment, the macrophages and dendritic cells stimulated by exosomes and immune complex cfDNA secreted significantly lower levels of cytokines including MIP-1a, MIP-1b, IL-4, IL-1β, IL-2, IL-1ra, MCP-1, IL-6, IL-9, IL-8, TNF-α and IFN-α. Compared with IgG enzyme treatment, DNASE1L3-treated exosomes and immune complex induced the lower level of cytokines secretion. The expression of CD80, CD86 and CD40 on the surfaces of macrophages and dendritic cells stimulated with exosomes and immune complexes pre-treated with DNASE1L3 enzyme were significantly reduced, but the expression of CD86 on the macrophages was slightly changed. The expression of CD80, CD86 and CD40 showed no difference in the two types of cells stimulated with exosomes and immune complexes pre-treated with IgG enzymes or none, but an overall downward trend existed indeed. Conclusion Exosomes and immune complex cfDNA increase significantly in the plasma of SLE patients, and they can stimulate strong responses of macrophages and dendritic cells.