The harmine derivatives were comprehensively investigated by computational tools to predict the structural, electronic, and spectroscopic properties as well as the chemical reactivity behavior. Physicochemical parameters showed that the harmine derivatives (H2 and H4) containing the -OH group at 2-position were more stabilized with the solvent dielectric constant than those of the other compounds (H1 and H3) including the -OCH3 substitution at 2-position. The PED analysis was used to assign the vibrational modes of all stable conformers of the harmine derivatives. TD-DFT simulations revealed that the lowest energy excitations were related to the H -> L transition, which was mainly characterized by n -> pi* for H1 and H2 compounds and pi -> pi* for H3 and H4 compounds. According to NBO analysis results, the highest contribution to the lowering of the molecular stabilization energy for all compounds was mainly due to the intramolecular charge transfer from the lone pair of the N atom as a donor orbital to pi* as an acceptor orbital. Global reactivity descriptors obtained from B3LYP/6-311++G(d,p) level implied that the trans-conformers of the studied compounds could be relatively more effective in their interaction with DNA, while the cis-conformers of them could be more eager to interact with the BSA molecule.