Objective To investigate the immunoprotective effect of a four-antigen fusion protein vaccine (referred to as 4 ×) composed of Toxoplasma gondii rhoptry protein 18 (ROP18), centrin family protein TGME49_237490, oocyst wall-specific protein ortholog TGME49_268230, and microneme protein 13 (MIC13). Methods The 4 × target gene was amplified using PCR assay, and recombinant plasmids were constructed and characterized. The successfully constructed plasmids were transformed into Escherichia coli BL21 competent cells for induced expression and affinity purification of the target protein, and the expression of the target protein was determined using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. Thirty-six female BALB/c mice were randomly divided into an immunization group and a control group, of 18 mice in each group. Mice in the immunization group were injected with 4 × protein and Freund’s adjuvant, and animals in the control group were administered with phosphate-buffered saline and Freund’s adjuvant. Immunizations were administered three times at two-week intervals. Blood samples were collected from the orbital sinus 0, 2, 4, 6 weeks following immunization, and serum samples were isolated. The levels of serum IgG antibodies were measured using ELISA. In the final detection, the levels of IgG antibody subtypes were also assessed. Two weeks after the final immunization, ten mice from each group were sacrificed, and spleens were aseptically harvested to prepare splenocyte suspensions. Splenocyte suspensions from five mice were used to measure cell proliferation using the CCK8 assay, while those from the other five mice were used for analysis of cytokine levels using flow cytometry. Mouse peripheral blood was sampled two weeks following final immunization, and proportions of mouse T cell subsets were detected using flow cytometry in five mice from each group. Eight mice were sampled from each group two weeks following final immunization and challenged intraperitoneally with 100 tachyzoites of the T. gondii RH strain. Then, the burdens of T. gondii in organs of 3 mice were examined using real-time quantitative fluorescence PCR (qPCR) assay, and the survival of other 5 mice were monitored daily and survival curves were plotted, statistical analysis was performed using GraphPad Prism 8 software. Results The PCR amplification product of the 4 × gene was 3 576 bp, which was consistent with the expected size. Recombinant plasmids showed expected band sizes upon characterization with PCR assay and enzymatic digestion. The target protein was successfully expressed and purified, with prominent bands observed at M_r 110 000 by both SDS-PAGE and Western blotting. ELISA results showed that the IgG antibody levels (A₄₅₀ values) in the immunized group at weeks 0, 2, 4, and 6 post-immunization were 0.079 ± 0.004, 0.759 ± 0.179, 1.670 ± 0.243, and 2.461 ± 0.056, respectively. The corresponding values for the control group were 0.080 ± 0.006, 0.067 ± 0.009, 0.080 ± 0.014, and 0.076 ± 0.011, respectively. Except for week 0 (t = 0.05, P > 0.05), the IgG levels in the immunized group were significantly higher than those in the control group (t = 9.88, 23.33, 34.66; all P < 0.05). At week 6 post-immunization, the absorbance values of IgG1 and IgG2a antibodies were 3.202 ± 0.401 and 3.725 ± 0.066 in the immunization group and 0.082 ± 0.003 and 0.059 ± 0.017 in the control group (t = 13.73, 96.83; both P < 0.05), respectively. Flow cytometry detected that the levels of IFN-γ, IL-2, IL-12, IL-4, and IL-10 were (4 998.52 ± 2 131.24), (5.76 ± 1.02), (1.38 ± 0.86), (28.83 ± 1.64), and (3 376.57 ± 218.48) pg/ml in the immunization group and (8.90 ± 0.17), (3.05 ± 0.50), (0.06 ± 0.13), (11.18 ± 1.58), (13.87 ± 3.55) pg/ml in the control group (t = 4.89, 4.61, 3.06, 15.93, 30.30; all P < 0.05), respectively, and the proliferation level of splenocytes was 3.286 ± 0.552 in the immunization group and 1.251 ± 0.157 in the control group (t = 9.15, P < 0.05). The proportions of CD3⁺CD4⁺ and CD3⁺CD8⁺ T cells were (31.15 ± 2.83)% and (18.06 ± 2.82)% in the immunization group and (26.82 ± 1.17)% and (9.33 ± 1.22)% in the control group (t = 3.16, 6.36; both P < 0.05), respectively. RT-qPCR assay detected that parasite burdens were (206.7 ± 16.0), (100.2 ± 15.4), and (7.1 ± 2.0) fg/mg in the mouse liver, spleen, and lungs in the immunization mice, and (20 086.0 ± 1 310.0), (26 254.0 ± 9 658.0) and (5 300.0 ± 741.2) fg/mg in the control group (t = 26.29, 4.69, 12.37; all P < 0.05). All mice in the control group died within 8 days following challenge, in the immunized group, only one mouse survived throughout the 30 d observation period, with a significantly prolonged survival time (χ² = 7.47, P < 0.05). Conclusion The four-antigen fusion protein vaccine may induce effective humoral and cellular immune responses in mice and present a significant protective effect against acute T. gondii infection.