Recent outbreaks of orf in China have been attributed to a novel strain of Orf virus (ORFV) designated ORFV-Jilin. Currently, monoclonal antibodies (Mabs) have not yet been developed against this specific pathogen even though such entities could have potential applications regarding the diagnosis and characterization of ORFV-Jilin. Therefore, the current study was undertaken to generate Mab against the immunodominant ORFV059 protein of this virus. For this purpose, the ORFV-Jilin ORFV059 protein was expressed in Escherichia coli and subsequently used as an antigen to immunize mice and for the initial screening of hybridomas prepared from the mice for their ability to produce anti-ORFV059 protein Mabs via an indirect ELISA. Ten, positive hybridomas were identified in this manner and verified based on the ability of their released Mab to react specifically with both naturally and artificially expressed ORFV059 protein in Western blots. The two hybridomas with the greatest propensity to secrete Mab were subcloned three times before being introduced intraperitoneally into mice. Afterwards, both Mab were separately purified from the mice's ascetic fluids and found to successfully recognize the ORFV-Jilin ORFV059 protein in a variety of immunological assays. Thus, the widespread utility of these Mab as a diagnostic core reagent should prove invaluable for further investigations regarding the mechanisms of orf pathogenesis and the control of this disease. In this regard, it should be noted that Mab A3 was used to confirm the predicted late expression of the ORFV-Jilin ORFV059 protein during virus replication.