Theophylline is a potent bronchodilator for the treatment of asthma, bronchitis, and emphysema. Its narrow therapeutic window (20–100 μM) demands that the blood concentration of theophylline be monitored carefully, which can be achieved by aptamer capture. Thus, an understanding of what occurs when aptamers bind to theophylline is critical for identifying a high-affinity and high-specificity aptamer, which improve the sensitivity and selectivity of theophylline detection. Consequently, there is an urgent need to develop a simple, convenient, and nondestructive method to monitor conformational changes during the binding process. Here, we report the determination of the affinity of a selected aptamer and theophylline via biolayer interferometry (BLI) experiments. Additionally, using surface-enhanced Raman spectroscopy (SERS), the conformational changes on theophylline–aptamer binding were identified from differences in the SER spectra. Finally, molecular dynamics (MD) simulations were used to identify the specific conformational changes of the aptamer during the binding process. Such a combined BLI-SERS-MD method provides an in-depth understanding of the theophylline–aptamer binding processes and a comprehensive explanation for conformational changes, which helps to select, design, and modify an aptamer with high affinity and specificity. It can also be used as a scheme for the study of other aptamer–ligand interactions, which can be applied to the detection, sensing, clinical diagnosis, and treatment of diseases. We established a method that synthesizes BLI experiments, SERS experiments, and MD simulations to analyze the conformational changes of aptamers in the process of binding to theophylline. This provides an in-depth understanding of theophylline–aptamer binding processes and a comprehensive explanation for conformational changes. Display omitted •Method integrates BLI, SERS, and MD simulations to study the aptamer’s conformational changes.•Method acquires a comprehensive description of the conformational changes during binding process.•Method provides strong support to increase the affinity and stability of other ligand–aptamer.