Grapevine fanleaf virus (GFLV) was isolated from symptomatic grapevine (Vitis vinifera L.) leaf samples obtained from El Kalubia Governorate. Three out or 25 samples were positive for GFLV by DAS-ELISA using specific polyclonal antibodies raised against purified GFLV preparation and five samples were positive by RT-PCR. RT-PCR detected GFLV in both fresh and dried tissues. A fragment (321 bp) of the coat protein gene of GFLV was amplified by reverse transcription-polymerase chain reaction (RT-PCR) using two primers specific for the coat protein gene or GFLV. Nucleotide sequences of the RT-PCR products confirmed that these sequences were amplified from the GFLV coat protein gene. A specific GFLV Dig labeled DNA probe was prepared by PCR and detected the GFLV virus in fresh leaves up to 10-5 dilution in dot blot hybridization assay. It was suggested that the inhibitory compounds released during the extraction of RNA constitute a limiting factor for the detection of GFLV in infected vines. Both immunological and molecular detection methods provide tools assisting in the understanding of the epidemiology and diversity of nepoviruses as well as to facilitate resistance breeding, cultivar selection, and development of control strategies.