Objective To explore the molecular interaction mechanism between theophylline and DNA aptamers (APT) using various techniques. Methods Firstly, biolayer interferometry (BLI) was employed to determine the affinity between 2 μmol/L APT and a gradient of theophylline concentrations (31.25, 62.5, 125, 250, 500 and 1 000 nmol/L) in 100 mmol/L Tris-HCl＋3 mmol/L MgCl₂ (pH 7.5). Then, circular dichroism (CD) was used to measure the CD spectra of 4 μmol/L APT before and after binding with 2 μmol/L theophylline. Finally, surface-enhanced Raman spectroscopy (SERS) was used to record spectra of 50 μmol/L APT and 200 μmol/L theophylline before and after mixing, and difference spectra were analyzed between the theophylline-APT complex and APT alone. Results The BLI experiment showed good affinity between APT and theophylline with an equilibrium dissociation constant K_d of 0.25 μmol/L. Moreover, the affinity of APT for theophylline was much higher than those of theobromine and caffeine, and the selectivity of APT for theophylline was good. With random DNA sequences as negative controls, it was confirmed that the binding between APT and theophylline was specific rather than non-specific adsorption. The CD experiment confirmed that the conformation of APT transformed to the stem-ring structure after binding to theophylline. The SERS experiment showed that the combination of APT and theophylline caused changes in guanine, adenine and cytosine on APT, indicating that the conformation of APT changed after binding to theophylline, which in turn led to changes in the SERS signal of APT. Conclusion Theophylline interacts with DNA APT molecules with good affinity, inducing changes in the molecular structure of APT and altering the positions of its bases.