CAR-T cell therapy using lentiviral vectors can lead to false-positive HIV RNA detection, making distinguishing true infection from vector-related signals challenging. A 64-year-old male with relapsed/refractory DLBCL (RR-DLBCL) underwent multiple lines of treatment, including R-CHOP, R-ICE, autologous stem cell transplantation (ASCT), and tisagenlecleucel (tisa-cel, Kymriah). Infectious disease screening before CAR-T therapy was negative for HIV. However, four months post-infusion, during evaluation for second-line CAR-T therapy targeted CD20, HIV RNA was detected in Roche Cobas HIV-1 assay targeted dual target, 5'LTR and gag gene (48 copies/mL). Serial testing showed persistent but low-level positivity of HIV RNA. Retrospective analysis of stored serum samples revealed HIV RNA negativity before tisa-cel infusion but positivity post-infusion in Roche Cobas HIV-1 assay. Additional testing using the Alinity m HIV-1 assay (dual target: 5'LTR and pol gene) and the Abbott RealTime HIV-1 assay (single-target: pol gene) confirmed that only the dual-target assay yielded positive results, suggesting lentiviral vector cross-reactivity rather than actual HIV infection. This case underscores the potential for false-positive HIV-1 RNA detection in CAR-T cell treatment recipients due to vector-derived sequences, emphasizing the need for cautious interpretation of HIV-1 testing.