The two-dimensional (2D) periodic Au nanosphere array with silver coating was prepared by using a colloidal monolayer template to obtain a Au nanosphere array and subsequently depositing silver thin coating on it, which could be used as an optical sensor to effectively detect H2S. Such periodic Au nanosphere array with silver coating displayed a surface plasmonic resonance (SPR) peak and an optical diffraction peak. Compared with the SPR peak, the diffraction peak, originated from the periodic arrangements of the obtained array, demonstrated a more sensitive optical change to detect H2S with a significant redshift as the H2S concentration increased. It was attributed to the increase of the refractive index of the environment around the Au nanosphere arrays with silver coating due to the partial formation of Ag2S after detecting H2S. Furthermore, the H2S sensor based on the change of the optical diffraction peak, showed an excellent selectivity and it was very sensitive to detect H2S from 2 to 30 μM. This method was investigated by the analysis in H2S-spiked blood samples, which indicates that the method has the potential to detect H2S in blood samples. The presented work provides a new strategy of utilizing the optical diffraction peak of the periodic array to develop promising sensors.