The effects of T-cell immunoglobulin mucin molecule-3 (Tim-3), transforming growth factor β (TGF-β), and chemokine-12 (CXCL12) expression on the prognosis of patients with diffuse large B-cell lymphoma (DLBCL) have not been elucidated. To examine the correlation between Tim-3, TGF-β and CXCL12 expression and DLBCL prognosis. Lymph node tissues of 97 patients with DLBCL and 93 normal-response hyperplastic lymph node tissues treated from January 2017 to May 2019 were selected as the DLBCL and control groups, respectively. The expression of Tim-3, TGF-β, and CXCL12 was detected immunohistochemically. Patients were followed up for 3 years, and progression-free survival was recorded. Cox multifactorial analysis was performed to analyze the risk factors for poor prognosis. The positive expression rates of Tim-3, TGF-β, and CXCL12 were higher in DLBCL tissues than in non-cancerous (control) tissues (P < 0.05). One-year post-surgery, the positive expression rates of Tim-3, TGF-β, and CXCL12 were higher in patients with effective treatment than in those with ineffective treatment (P < 0.05). The 3-year progression-free survival of 97 patients with DLBCL was 67.01% (65/97). Univariate analysis revealed that clinical stage, bone marrow infiltration, International Prognostic Index (IPI) score, Tim-3 positivity, TGF-β positivity, and CXCL12 positivity were associated with poor prognosis (P < 0.05). Multivariate Cox regression analysis demonstrated that clinical stage III-IV, bone marrow infiltration, mediate-to-high-risk IPI scores, Tim-3 positivity, TGF-β positivity, and CXCL12 positivity were independent risk factors affecting prognosis (P < 0.05). DLBCL tissues exhibit high positive expression of Tim-3, TGF-β, and CXCL12, and a high expression of all three indicates a poor prognosis.