Infection is a significant cause of morbidity and mortality in patients with chronic kidney disease, especially who were under dialysis due to their depressed immunity. Toxoplasma gondii is a ubiquitous parasite that causes severe manifestations in immunocompromised patients. This case-control study was conducted to the immunodiagnosis and molecular validation of T. gondii infection among patients with end-stage renal disease undergoing haemodialysis. The study population consisted of 260 haemodialysis patients and 259 healthy controls referred to the main dialysis centres of Tehran, Iran during 2016. Anti-T. gondii immunoglobulin G (IgG) and immunoglobulin M (IgM) antibodies were assessed using enzyme-linked immunosorbent assay. As well, the T. gondii genomic DNA in whole blood samples of IgM-positive patients and healthy controls was evaluated using GRA6-polymerase chain reaction (PCR) and SAG1-loop-mediated isothermal amplification (LAMP) assays. The anti-T. gondii IgG and IgM antibodies were detected in 175 (67.3%) and 18 (7%) of haemodialysis patients and 122 (47%) and 4 (1.5%) of controls, respectively. Two of the 18 blood samples from IgM-positive patients and none of the IgM-positive control subjects were positive by GRA6-PCR. Whereas, nine and two blood samples of IgM-positive patients and controls were positive for Toxoplasma DNA by a SAG1-LAMP technique respectively. The seropositivity of the Toxoplasma IgM antibody was significantly different between haemodialysis patients and healthy controls which was confirmed by PCR and LAMP. The higher prevalence of T. gondii infection in haemodialysis patients compared with the controls proposes that these patients can be a group at risk for toxoplasmosis and screening for toxoplasmosis before dialysis is necessary for the patients.