An acrylamide biosensor was developed by utilizing purine bases, i.e. guanine and adenine, through computational and electrochemical approaches. The molecular docking simulation proved that interaction of double-stranded DNA with the purine bases has the lowest Gibbs binding free energy compared to other biomolecules with a ΔGbinding of −4.2759 kcal/mol. Meanwhile, cyclic voltammetry of both guanine and adenine in 0.1 M phosphate buffer solution at pH 7.4 using a boron-doped diamond electrode showed an irreversible oxidation peak in the potential range of 0 to +1.8 V (vs. Ag/AgCl), confirming that the oxidation reaction was irreversible. The current of these peaks decreased linearly with the concentration of acrylamide due to the adduct formation between the purine bases and acrylamide. The formation of acrylamide adducts between acrylamide and purine bases was confirmed by the shift of the peak wavelength of the UV spectrum from 260 to 257 nm. The use of guanine for acrylamide sensing showed a linear calibration curve in the concentration range of 0.20–1.00 µM (R2 = 0.99) with a limit of detection and limit of quantification attained at 0.11 and 0.36 µM, respectively. In the case of adenine, a linear calibration curve was observed in the concentration range of 0.14–1.00 µM (R2 = 0.99) with a limit of detection and limit of quantification of 0.10 and 0.34 µM, respectively. The developed method was successfully performed for the acrylamide determination in coffee samples and was validated by HPLC.