Cancer disease is developing all over the world mainly in developing countries. We should learn more about DNA–ligand interactions to design new drugs that target biological activities like transcription, replication and translation of particular genes. To understand the mechanism of action and design-specific DNA binders, the evaluation of DNA–ligand interactions is critical. Novel barbituric acid derivatives based on (benzyloxy)benzaldehydes were synthesized and evaluated as DNA-binding agents. Among products, molecular docking studies revealed that 4j and 4m have the best interactions with the ctDNA via the minor groove binding. These results were approved by the quantum mechanics calculations. The interaction profiles of the selected compound ( 4j and 4m ) with DNA were evaluated by UV–Visible titration. UV–Visible titration data confirm this interaction. According to the molecular modeling results, the Structure–Activity relationships for all synthesized barbituric acid derivatives were proposed. It was observed that N,N -dimethyl barbituric acid/4-hydroxybenzaldehyde derivatives have better DNA interactions than barbituric acid/vanillin and barbituric acid/3-hydroxybenzaldehyde derivatives. Supplementary Information The online version contains supplementary material available at 10.1007/s13738-022-02576-x.