Interferon-gamma (IFN-γ) is a glycoprotein generated by lymphocytes that possesses anti-tumor, antiviral and immunomodulatory functions. IFN-γ assays are broadly employed in immunological research and clinical diagnostic tests. Intracellular IFN-γ staining, in particular, is an important immune assay that allows simultaneous determination of cellular phenotype and antigen-specific T cell response. Aptamers have great potential for molecule detection and can bind to target molecules with high affinity and specificity. In this study, a novel 59-mer DNA aptamer (B1-4) was developed for assay of intracellular IFN-γ. The selected aptamer bound to IFN-γ with a Kd of 74.5 nM, with minimal cross-reactivity to albumin. The aptamer was also found capable of binding with paraformaldehyde-fixed IFN-γ. Moreover, B1-4 could enter permeated and paraformaldehyde-fixed lymphocytes, and bound to intracellular IFN-γ produced by these cells. When FITC-labeled B1-4 was used to stain a group of lymphocytes, the average fluorescence of the cells was positively correlated with the number of PMA-stimulated lymphocytes within the group. A standard curve could thus be established for assessing the fraction of IFN-γ-producing cells in a cluster of lymphocytes. The selected aptamer hence provides a novel approach for assaying intracellular IFN-γ generated by a group of lymphocytes, and may have application potential in both scientific research and clinical laboratory test.