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Bovine herpesvirus-1 is highly contagious virus of cattle and buffaloes all over the world. It establishes lifelong latency in ganglionic neurons of the peripheral nervous system. Since, trigeminal ganglia are the main sites of latency, therefore, it is challenging to detect BoHV-1 in latently infected live animals. No research work has been done to correlate the sero-prevalence and latency in peripheral blood mononuclear cells (PBMC). The present study was designed to detect BoHV-1 latency related transcript or microRNA in peripheral blood mononuclear cells of sero-positive animals. The highly sensitive RT-qPCR assays based on TaqMan chemistry have been developed for the detection of transcripts of BoHV-1 latency. The limit of detection (LOD) of the assays for ORF-1 specific RT-qPCR and miRNA specific RT-qPCR was 460 copies and 117 copies respectively. The efficiency of the developed assays was 93.5% for ORF-1 and 97.93% for miRNA transcript. None of the PBMC samples of seropositive animals found positive for ORF-1 and miRNA transcripts in developed assays.The absence of latency specific transcripts in PBMC might be due to very low expression i.e. beyond the LOD of newly developed assays or absence of latency in PBMC of seropositive animal. However, further studies are required to establish the fact. To the best of authors knowledge, this is the first report of latency-specific RT-qPCR assay development and its application in PBMC of BoHV-1 seropositive cattle.