An electrochemical aptasensor for theophylline detection was developed by integrating computational analysis, gold nanoparticle–modified screen-printed carbon electrodes (SPCE/AuNP), and hydrogel-forming microneedles. Molecular docking and molecular dynamics simulations confirmed the preferential and stable binding of the DNA aptamer to theophylline within a conserved stem–loop region, providing a molecular basis for selectivity. AuNP modification significantly enhanced electron-transfer kinetics and electroactive surface area, enabling sensitive signal transduction. The aptasensor exhibited a linear response over the range of 10–1000 μM with a detection limit of 0.39 μM and high reproducibility (RSD 1.59 %). Excellent selectivity was observed against common interferents. Integration with hydrogel microneedles enabled the detection of theophylline in spiked human blood, achieving a detection limit of 0.29 μM and satisfactory recovery. These results demonstrate the potential of the developed microneedle-assisted aptasensor as a minimally invasive platform for therapeutic drug monitoring of theophylline. © 2026 Vietnam National University, Hanoi.