Abstract Background Blastocystis hominis (Bh) is zoonotic parasitic pathogen with a high prevalent globally, causing opportunistic infections and diarrhea disease. Human immunodeficiency virus (HIV) infection disrupts the immune system by depleting CD4⁺ T lymphocyte (CD4⁺ T) cell counts, thereby increasing Bh infection risk among persons living with HIV (PLWH). However, the precise association between Bh infection risk and HIV-related biological markers and treatment processes remains poorly understood. Hence, the purpose of the study was to explore the association between Bh infection risk and CD4⁺ T cell counts, HIV viral load (VL), and duration of interruption in antiviral therapy among PLWH. Methods A large-scale multi-center cross-sectional study was conducted in China from June 2020 to December 2022. The genetic presence of Bh in fecal samples was detected by real-time fluorescence quantitative polymerase chain reaction, the CD4⁺ T cell counts in venous blood was measured using flowcytometry, and the HIV VL in serum was quantified using fluorescence-based instruments. Restricted cubic spline (RCS) was applied to assess the non-linear association between Bh infection risk and CD4⁺ T cell counts, HIV VL, and duration of interruption in highly active antiretroviral therapy (HARRT). Results A total of 1245 PLWH were enrolled in the study, the average age of PLWH was 43 years [interquartile range (IQR): 33, 52], with 452 (36.3%) being female, 50.4% (n = 628) had no immunosuppression (CD4⁺ T cell counts > 500 cells/μl), and 78.1% (n = 972) achieved full virological suppression (HIV VL < 50 copies/ml). Approximately 10.5% (n = 131) of PLWH had interruption. The prevalence of Bh was found to be 4.9% [95% confidence interval (CI): 3.8–6.4%] among PLWH. Significant nonlinear associations were observed between the Bh infection risk and CD4⁺ T cell counts (P_{for nonlinearity} < 0.001, L-shaped), HIV VL (P_{for nonlinearity} < 0.001, inverted U-shaped), and duration of interruption in HARRT (P_{for nonlinearity} < 0.001, inverted U-shaped). Conclusions The study revealed that VL was a better predictor of Bh infection than CD4⁺ T cell counts. It is crucial to consider the simultaneous surveillance of HIV VL and CD4⁺ T cell counts in PLWH in the regions with high level of socioeconomic development. The integrated approach can offer more comprehensive and accurate understanding in the aspects of Bh infection and other opportunistic infections, the efficacy of therapeutic drugs, and the assessment of preventive and control strategies. Graphical Abstract