Although materials and engineered surfaces are broadly utilized in creating assays and devices with wide applications in diagnostics, preservation of these immuno-functionalized surfaces on microfluidic devices remains a significant challenge to create reliable repeatable assays that would facilitate patient care in resource-constrained settings at the point-of-care (POC), where reliable electricity and refrigeration are lacking. To address this challenge, we present an innovative approach to stabilize surfaces on-chip with multiple layers of immunochemistry. The functionality of microfluidic devices using the presented method is evaluated at room temperature for up to 6-month shelf life. We integrated the preserved microfluidic devices with a lensless complementary metal oxide semiconductor (CMOS) imaging platform to count CD4(+) T cells from a drop of unprocessed whole blood targeting applications at the POC such as HIV management and monitoring. The developed immunochemistry stabilization method can potentially be applied broadly to other diagnostic immuno-assays such as viral load measurements, chemotherapy monitoring, and biomarker detection for cancer patients at the POC.