The rhoptry kinase 18 of Toxoplasma gondii (TgROP18) has been identified as a key virulence factor that allows the parasite to escape from host immune defences and promotes its proliferation in host cells. Although much research is focused on the interaction between host cells and TgROP18, the development of monoclonal antibodies (mAbs) against TgROP18 has not been reported till date. To produce mAbs targeting TgROP18, two hybridomas secreting mAbs against TgROP18, designated as A1 and T2, were generated using cell fusion technology. The subtypes of the A1 and T2 mAbs were identified as IgG3 λ and IgM κ, and peptide scanning revealed that the core sequences of the antigenic epitopes were 180LRAQRRRSELVFE192 and 351NYFLLMMRAEADM363, respectively. The T2 mAb specifically reacted with both T. gondii type I and Chinese I, but not with T. gondii type II, Plasmodium falciparum or Schistosoma japonicum. Finally, the sequences of heavy chain and light chain complementarity-determining regions of T2 were amplified, cloned and characterized, making the modification of the mAb feasible in the future. The development of mAbs against TgROP18 would aid the investigation of the molecular mechanisms underlying the modulation of host cellular functions by TgROP18, and in the development of strategies to diagnose and treat Toxoplasmosis.