HIV has caused a global pandemic over the last three decades. There is an unmet need to develop point-of-care (POC) viral load diagnostics to initiate and monitor antiretroviral treatment in resource-constrained settings. Particularly, geographical distribution of HIV subtypes poses significant challenges for POC immunoassays. Here, we demonstrated a microfluidic device that can effectively capture various subtypes of HIV particles through anti-gp120 antibodies, which were immobilized on the microchannel surface. We first optimized an antibody immobilization process using fluorescent antibodies, quantum dot staining and AFM studies. The results showed that anti-gp120 antibodies were immobilized on the microchannel surface with an elevated antibody density and uniform antibody orientation using a Protein G-based surface chemistry. Further, RT-qPCR analysis showed that HIV particles of subtypes A, B and C were captured repeatably with high efficiencies of 77.2 ± 13.2%, 82.1 ± 18.8, and 80.9 ± 14.0% from culture supernatant, and 73.2 ± 13.6, 74.4 ± 14.6 and 78.3 ± 13.3% from spiked whole blood at a viral load of 1000 copies per mL, respectively. HIV particles of subtypes A, B and C were captured with high efficiencies of 81.8 ± 9.4%, 72.5 ± 18.7, and 87.8 ± 3.2% from culture supernatant, and 74.6 ± 12.9, 75.5 ± 6.7 and 69.7 ± 9.5% from spiked whole blood at a viral load of 10,000 copies per mL, respectively. The presented immuno-sensing device enables the development of POC on-chip technologies to monitor viral load and guide antiretroviral treatment (ART) in resource-constrained settings.