Objective: Double-negative (DN) CD3+CD4–CD8– T lymphocytes represent a rare subset of unconventional peripheral T cells, accounting for only 3–5% of circulating T lymphocytes. Despite their low abundance, they play an important role in the pathogenesis of inflammation, cancer, autoimmune diseases, and allergic asthma. However, the cytokine profile of DN T cells remains poorly studied. This work aimed to comprehensively characterize the spectrum of cytokines, chemokines, and growth factors secreted by DN T cells. Methods: DN T cells were isolated from in vitro culture of human peripheral blood mononuclear cells (PBMCs). Cells were activated using anti-CD3/CD28 magnetic beads. The cytokine profile was assessed by multiplex analysis using xMAP technology, allowing simultaneous quantification of 48 analytes, including cytokines, chemokines, and growth factors. Results and Discussion: The pro-inflammatory αβTCR+CD161+ DN T cell subset exhibited a distinct secretory profile. Elevated levels of pro-inflammatory cytokines (TNFα, IFNγ, IL-3, IL-13, IL-27), chemokines (IL-8/CXCL8, MIG/CXCL9, MIP-1α/CCL3, MIP-1β/CCL4, RANTES/CCL5), and growth factors (M-CSF, GM-CSF) were detected. These findings suggest that DN T cells possess a broad and functionally diverse cytokine secretion potential, implicating them in a wide range of immune processes. Conclusions: The absence of regulatory cytokines and the predominant production of inflammatory mediators suggest their potential role in immune activation rather than suppression. These findings provide a basis for further investigation into the functional heterogeneity and therapeutic targeting of DN T cells in autoimmune and inflammatory diseases.