Purpose This study aimed to explore the prevalence of Toxoplasma gondii (T. gondii) among patients in Guangzhou city, South China, and to identify susceptible patient populations and analyze the causes of infection differences. Methods From May 2020 to May 2022, a total of 637 sera were collected from patients, and 205 sera were collected from health participants as health control. All sera were examined by colloidal gold kits to detect the positivity of antibodies against T. gondii. And the positivity of antibodies in sera was confirmed with ARCHITECT i2000SR system. Results The prevalence of T. gondii infection in patients was 7.06% (45/637), which was lower than the prevalence in health participants 4.88% (10/205). Among patients, 34 (5.34%) were positive only for IgG, 10 (1.57%) were only for IgM, and 1 (0.16%) was positive for both IgG and IgM. There was a significant difference in prevalence between male and female patients, but not among different age groups or diseases groups. The prevalence of T. gondii infection in diseases groups varied. The prevalence was relatively high in patients with the disorders of thyroid gland and the malignant neoplasms of digestive organs, which suggests that caution should be taken to avoid T. gondii infection in these patients. Surprisingly, the prevalence was quite low in diffuse Large B-cell Lymphoma (DLBC) patients. This may be due to the overexpression of TNF-α in tumor tissues of DLBC patients and the higher protein level of TNF-α in sera of DLBC patients. Conclusion This study provides a systematic exploration of the prevalence of T. gondii infection in patients in a tertiary hospital. Our data contributes to a better understanding of the epidemic investigation of T. gondii among patients in South China, which can help the prevention and treatment of the disease caused by T. gondii infection.