The new diazo ligand, and its complexes with metals ions Cu(II), Zn(II), Cd(II), and Ag(I), were synthesized, and ligand synthesis was done via diazotization of 4,4'-Methylenedianiline (MDA), and then coupling was carried out with aniline derivative in good yields by using spectroscopic techniques such as FT-IR, UV-Visible, NMR, and elemental analysis for characterization. Also, the analytical measurements were done such as the detection of conditions of the optimal reaction (reagent concentration, pH, etc.), the detection limit, linearity, and sensitivity. Moreover, the biological activity of the synthesized diazo ligand and its complexes were tested against four types of bacteria (Staphylococcus aureus, Enterococcus feacalis, E.coli, and Pseudomonas aeroginosa,) in vitro. They showed the promising biological activity toward these examined organisms.