Graphene oxide modified with aqueous ammonia samples were prepared by impregnation method, and the catalytic performances of the as-prepared samples in Knoevenagel condensation reaction of benzaldehyde with malononitrile were investigated. The properties of the catalysts were characterized by X-ray diffraction, Raman spectra, Fourier transform infrared spectroscopy, NH3-TPD and elemental analysis. Results indicated that graphene oxide modified with aqueous ammonia samples exhibited excellent performances in the Knoevenagel condensation reaction of benzaldehyde with malononitrile. The catalytic activity increased gradually with increasing the concentration of aqueous ammonia. A 93.6% conversion of benzaldehyde and a 94.8% selectivity of benzylidene malononitrile was obtained over graphene oxide modified with 5 wt% aqueous ammonia in the appropriate conditions. It is very important that the graphene oxide modified with aqueous ammonia samples can be reused for 5 times only by simple filtration after reaction. Characterization results indicated that graphene oxide modified by ammonia can effectively immobilize NH4+ on the surface of graphene oxide.