A Real-Time RT-PCR (Reverse Transcription-Polymerase Chain Reaction) TaqMan® assay was used for the specific detection and absolute quantification of the Grapevine fanleaf virus (GFLV) in the infected tissues. The assay was targeted to a conserved region located in the 2BMP (movement protein) gene of the GFLV RNA2 molecule. This method was successfully used for the detection and quantification of GFLV in infected grapevines from South Moravia which were evaluated three times in 2011 during major phenophases. Moreover, DAS-ELISA detection was applied for the same samples to verify the higher sensitivity and reliability of Real-Time RT-PCR TaqMan®. The end-point dilution of ELISA was determined to calculate the detection limit of this method for GFLV. The Real-Time RT-PCR TaqMan® assay detected GFLV in all eight of the Moravian GFLV isolates tested, while DAS-ELISA was unable to detect GFLV in four of them. Isolate HV5 was easily detected by ELISA in 2004, when it was collected in the field. In the current experiment, after several years of cultivation in a screenhouse, the concentration of GFLV decreased under the detection limit of ELISA. The detection limit, calculated as the amount of viral RNA2 molecules present in a tested sample, for GFLV detection using Real-Time RT-PCR TaqMan® was approximately 1000-fold higher than that of ELISA which makes the method suitable for certification of grapevine propagation materials. GFLV detection was possible all the year, using phloem tissues.