Preimplantation bovine blastocyst supernatants exhibit sex-dependent antiviral activity, due to the ruminant pregnancy recognition signal interferon tau (IFNT). Differing potencies of IFNT variants have been supposed as cause, although evidence remains scarce. Here, we aimed at quantifying the sex-dependent IFNT production on transcriptional, translational and biological activity level in bovine blastocysts, to elucidate the origin of differences in antiviral activity between male and female blastocysts. Day 8 bovine blastocysts were co-cultured with endometrial stroma cells for 48 h. The embryonic IFNT mRNA expression was determined by quantitative reverse transcription followed by polymerase chain reaction (RT-qPCR). Additionally, the IFNT protein concentration was determined using a sensitive in-house developed IFNT-specific enzyme-linked immunosorbent assay (ELISA). The biological activity was assessed by quantifying the response of interferon-stimulated gene (ISG) expression in endometrial stroma cells. While the IFNT-specific ELISA displayed a limit of detection of 7.3 pg/mL, the stroma cell culture system showed to react to as little as 0.1 pg/mL IFNT in RT-qPCR analysis. The female blastocysts had a significant, 5.6-fold, 3.6-fold and 5.2-fold higher IFNT production than male blastocysts as determined by transcript abundance, protein concentration and, protein activity, respectively. Additionally, all parameters correlated positively, and therefore, we conclude that female blastocysts most likely have an increased IFNT gene and protein expression rather than expressing more potent IFNT variants.