A novel bis-Schiff base of 5,5′-methylene-bis(salicylaldehyde)-5-methyl-2-[(2-aminophenyl) amino]thiophene-3-carbonitrile was synthesized with 5,5′-methylene-bis(salicylaldehyde) and 5-methyl-2-[(2-aminophenyl)amino]thiophene-3-carbonitrile as raw materials, and the 5,5′-methylene-bis(salicylaldehyde) was derived from salicylaldehyde and trioxane. The results of elemental analysis and mass spectrometry confirmed that the molecular formula of Schiff base was C39H30N6O2S2. The structure of the Schiff base was characterized by FT-IR and 1HNMR. The antibacterial activities of the Schiff base were screened by the Oxford cup agar diffusion method. The experimental results showed that the Schiff base had certain antibacterial activities against four bacteria and the diameter of the inhibition zone was 15.0 mm against Escherichia coli with the concentration of 1.5 mg•mL-1. The antibacterial activities of the sequence were Escherichia coli, Bacillus subtilis, Staphylococcus aureus and Staphylococcus epidermidis, respectively.