Abstract: OBJECTIVE To investigate the diagnostic value of serum soluble T-cell immunoglobulin mucin molecule 3 (sTim-3) and FMS-like tyrosine kinase 3 ligand (Flt3L) for human immunodeficiency virus (HIV)/hepatitis B virus (HBV) coinfection. METHODS A retrospective study was conducted on 138 HIV-infected patients (HIV group), 159 HBV-infected patients (HBV group) and 123 HIV/HBV coinfected patients (HIV/HBV group) admitted to Shanxi Province Fenyang Hospital and the First Hospital of Shanxi Medical University from Jan. 2019 to Mar. 2025, with 260 healthy individuals examined during the same period as controls. Serum sTim-3 and Flt3L levels were measured by enzyme-linked immunosorbent assay (ELISA). Receiver operating characteristic (ROC) curves were plotted to evaluate the diagnostic efficacy of sTim-3 and Flt3L alone or in combination for HIV/HBV coinfection. A restricted cubic spline (RCS) model was used to analyze the dose-response relationship between sTim-3, Flt3L and HIV/HBV coinfection. RESULTS The levels of sTim-3, Flt3L, lactate dehydrogenase (LDH), aspartate aminotransferase (AST), alanine aminotransferase (ALT), γ-glutamyl transferase (GGT) and total bilirubin (TBIL) in the HBV, HIV and HIV/HBV groups were higher than those in the control group (P<0.05). The HIV/HBV group exhibited higher levels of sTim-3, Flt3L, LDH, ALT, AST, GGT and TBIL than the HBV and HIV groups (P<0.05). ROC analysis indicated that sTim-3, Flt3L and their combined detection had good diagnostic value for coinfection, with superior performance in combined diagnosis. RCS showed an increased risk of infection when sTim-3 >6.29 ng/ml and Flt3L >90.58 pg/ml (P<0.05). CONCLUSION Serum sTim-3 and Flt3L levels are significantly elevated in HIV/HBV coinfection, and their combined detection demonstrates excellent diagnostic value, serving as novel biomarkers for early diagnosis of coinfection.