In humans, there are three members of type III interferon family, designated as IFN-λ1 (IL-29), IFN-λ2 (IL-28A) and IFN-λ3 (IL-28B). Bacterial infections, including Mycobacterium tuberculosis infection, trigger the expression of interferon I and II genes, but little is known about their effect on IFN-λ genes. It has been found that M. tuberculosis infection in A549 lung epithelial cells stimulate upregulation of IFN-λ2 genes in vitro. The aim of this study was to determine the IFN-λ2 levels in sputum of patients with active pulmonary tuberculosis comparing with levels in healthy persons sputum and sputum of persons with latent tuberculosis. IFN-λ2 level in the samples was quantified with sandwich enzyme-linked immunoassay kit for quantization of human IFN-λ2 (IL-28A). The IFN-λ2 level in sputum of patients with pulmonary tuberculosis was significantly higher comparing with IFN-λ2 levels in healthy group (P = 0.001) and latent tuberculosis group (P = 0.005). Logistic regression analysis has shown that IFN-λ2 level in sputum is related to culture result (OR = 4.14; 95% CI = 1.28–17.5; P = 0.019). There was the correlation between Bartlett score and IFN-λ2 levels in group with pulmonary tuberculosis (r = 0.641; P < 0.01). In conclusion, we have found that the inflammatory cells in sputum can be source of IFN-λ after receptor stimulation by M. tuberculosis components.