Introduction Co-infection with tuberculosis (TB) is the leading cause of death in individuals infected with human immunodeficiency virus (HIV)-1. Dolutegravir and lamivudine (DTG+3TC) has recently been recommended as the preferred first-line regimen for the treatment of new and treatment-experienced HIV-infected patients. The primary objective of this study was to determine the efficacy and safety of DTG 50 mg + 3TC 300 mg in HIV-positive antiretroviral therapy (ART)-naïve patients with TB who were receiving a rifampicin- or rifabutin-based treatment regimen, and to characterize viral suppression rates at week 48. Methods A single-center retrospective observational case series, spanning January 1, 2021 to March 1, 2023, was conducted in Guiyang Public Health Treatment Center. The outcomes of interest were successful TB treatment, viral load suppression, and immunological and biochemical indexes. Results All PWH had at least 48 weeks of follow-up, and all TB treatments were successful. A total of seven PWH (100%) achieved viral suppression (VL <50 copies/mL) from a baseline VL greater than 500,000 copies/mL. Among the PWH who started DTG+3TC after the initiation of the rifabutin-based anti-TB regimen, all achieved viral suppression by week 24, except for the non-suppressed PWH. CD4+ T-cell counts were greatly improved after antiretroviral treatment. The CD4+/CD8+ ratio increased by 0.38 (P < 0.001). Serum creatinine, total cholesterol, high-density lipoprotein cholesterol, and low-density lipoprotein cholesterol levels significantly increased (P = 0.054, P = 0.015, P < 0.001, and P < 0.05, respectively). There were no significant changes in body weight, alanine aminotransferase, aspartate aminotransferase, chronic kidney disease epidemiology collaboration-based serum creatinine, or triglyceride levels from baseline to week 48 (P > 0.05). No serious adverse events were observed. Conclusion This case series preliminarily validated the efficacy of DTG+3TC when combined with rifabutin-based anti-TB regimens in patients with TB and HIV.