Toxoplasma gondii, an opportunistic protozoan, infects one-third of people worldwide and could lead to serious outcomes in immunodeficient or immunocompromised populations. The present study aimed to investigate the prevalence and risk factors for T. gondii infection among high-risk populations in Jiangsu Province, eastern China. We conducted a cross-sectional survey among 4 categories of populations in 13 prefectures including HIV/AIDS patients, livestock breeding/processing (B/P) staff, pregnant women, and cancer patients. We detected specific immunoglobulin G and M (IgG and IgM) levels for each participant using enzyme-linked immunosorbent assay (ELISA) and asked to complete a questionnaire for each participant that covered sociodemographic information as well as the basic knowledge of attitudes toward and the practices for the prevention of toxoplasmosis. A total of 5231 participants distributed across 13 prefecture-level cities was surveyed, including 2455 males and 2776 females. Total seropositivity rate in each population category was as follows: 9.08% (HIV/AIDS patients), 11.65% (livestock B/P staff), 5.50% (pregnant women), and 12.89% (cancer patients). We detected IgM positivity in HIV/AIDS patients (0.47%, 6/1289), livestock B/P staff (0.08%, 1/1330), and cancer patients (0.46%, 6/1303) but not in pregnant women. Further, we detected IgM+IgG positivity only in cancer patients (0.31%, 4/1303). The seropositivity rate for pregnant women was significantly lower, while cancer patients were significantly higher. Higher educational levels were associated with lower seropositivity rates for T. gondii infection. High seropositivity rates were associated with long period of HIV infection among HIV/AIDS patients, frequent contact with livestock among livestock breeding/processing staff and male older patients among cancer patients, respectively. Analysis of practices across all participants showed that frequent contact with pets in everyday life or using the same cutting board for both raw and cooked foods leads to higher seropositivity rates. Therefore, we obtained the seroprevalence and risk factors of toxoplasmosis among high-risk populations in Jiangsu Province which could provide evidence for the implementation of control measures in the near future.