The aim of the present study was to determine seroprevalence of Toxoplasma gondii infectionin stray cats and correlation with oocyst shedding and IFN-γ concentration. From April to August 2016, one hundred fifty-nine stray cats were captured from various localities in Mashhad area. The blood and fecal samples were collected from each cat. The serum samples were examined to detect antibodies against T. gondii infection by ELISA assay and the fecal samples were microscopically examined for T. gondii oocyst detection. The concentration changes of IFN-γ in serum samples of seropositive and seronegative cats were measured using ELISA kit. The results showed that59.12% (94/159) of cats had antibodies against T. gondii infection. The seroprevalence of T. gondii infection in the adult cats above three years olds was higher than other groups. Regarding gender, month and region factors, the difference of seroprevalence of T. gondii infection was not significant. In this study, the Toxoplasma/Hammondia like oocyst (THLO) were detected in 2.56% (4/156) in fecal samples of one seropositive and three seronegative cats. Results also showed that the mean value for IFN-γ concentration in the seropositive cats was significantly higher than that of the seronegative cats. Based on the results, the high percentages of stray cats were infected with T. gondii in this area. The IFN-γ concentration of seropositive cats was higher than that of the seronegative cats.