Background <p>Opportunistic infections (OIs) remain a major clinical challenge in people living with HIV/AIDS (PLWHA), and CD4⁺ T-cell counts alone may not fully reflect immune dysfunction. This study aimed to establish predictive cut-off values for lymphocyte subsets to improve the assessment of OIs risk in PLWHA.</p> Methods <p>Peripheral blood samples were collected to assess lymphocyte subsets using flow cytometry between August 17, 2021, and April 19, 2023. Correlation analysis was performed to identify lymphocyte subset indicators strongly associated with CD4⁺ T-cell counts. Receiver operating characteristic (ROC) analysis was then performed to determine cut-off values for predicting OIs. Finally, the performance of cut-off values was tested in hospitalized PLWHA.</p> Results <p>Even when CD4 cell counts were above 200 cells/µl or close to normal, patients with abnormal immunological phenotypes had a higher prevalence of OIs. Other lymphocyte subsets have a high positive correlation with CD4⁺ T-cell counts, with the exception of CD3⁺CD8⁺/CD3⁺, CD3⁺HLA-DR⁺/CD3⁺, and CD3⁺CD8⁺HLA-DR⁺/CD8⁺, which are strongly negatively correlated. The correlation coefficients of CD4⁺CD28⁺ cells were particularly close to 1.0. Furthermore, CD4⁺CD28⁺ T lymphocytes demonstrated an AUC of 0.819 with a cut-off of 129 cells/µl, second only to CD4⁺ T cells in terms of AUC. Furthermore, the AUCs for CD3⁺CD4⁺CD45RA⁺, CD3⁺CD4⁺CD45RO⁺, CD3⁺CD4⁺CD25⁺CD127low, CD45RA⁺CD3⁺CD4⁺CD25⁺CD127low, and CD45RO⁺CD3⁺CD4⁺CD25⁺CD127low T cells were all &gt; 0.800, with sensitivities exceeding 90%.</p> Conclusion <p>In addition to lymphocyte counts, abnormal lymphocyte phenotype and immune activation status are strongly associated with OIs. The proposed cut-off values for selected lymphocyte subsets may help identify hospitalized PLWHA at increased risk of OIs.</p>