In this paper, a new series of substituted 1, 2,4-triazines was prepared by reacting 4-amino sulfanilic acid with substituted benzaldehyde to form the corresponding Schiff base compounds (1-5), and then reacting two moles of O-methoxy benzaldehyde with hydrazine to form a compound (6) and these compounds reacted with Schiff base (1-5) by applying the Diels-alder reaction in the presence of benzene as a solvent and refluxed it to cyclized into substituted of 1,2,4- triazines derivatives (7-11). Depending on the physical and spectroscopic properties of the prepared compounds that were identified, infrared spectroscopy and nuclear magnetic resonance were used. An evaluation of the biological activity of the prepared compounds was conducted and it gave anti-bacterial results, such as gram-positive and gram-negative when compared with known antibiotics.