Valganciclovir (VGCV) is primarily utilized in the management of cytomegalovirus (CMV) retinitis associated with AIDS and in the prevention of CMV transmission and illness among individuals undergoing organ transplantation. This study aimed to create nanomaterial-enhanced porous interfaces through a molecular imprinting strategy to facilitate the selective detection of VGCV. The sensor was fabricated via a photopolymerization technique on a glassy carbon electrode (GCE), employing VGCV as the template and 2-acrylamido-2-methyl-1-propane sulfonic acid (AMPS) as the functional monomer. The incorporation of zinc oxide nanoparticles (ZnO NPs) substantially enhanced the active surface area and porosity of the sensor. Comprehensive electrochemical and morphological assessments of the VGCV/AMPS/ZnO NPs/MIP-GCE sensor were performed utilizing scanning electron microscopy, cyclic voltammetry, and electrochemical impedance spectroscopy. The sensor demonstrated the capability to detect VGCV within a concentration ranging from 1 to 20 pM through an indirect measurement method. The calculated limits of detection for standard and serum samples were 2.89 × 10− 13 M and 1.99 × 10− 13 M, respectively, underscoring the sensor’s sensitivity and practicality. The imprinting factor (IF) investigation was conducted on a few medications that are structurally comparable to VGCV, revealing relative IF values that confirmed the sensor’s selectivity for VGCV. To further comprehend the interactions between the template and functional monomer, density functional theory computations were used. These theoretical analyses not only supported the adjustment of the template and monomer ratio experimentally but also revealed how these interactions change in the presence of ZnO NPs, allowing for the evaluation of their impact on binding energies.