Alloy nanostructures unveil extraordinary plasmonic phenomena that supersede the mono-metallic counterparts. Here we report silver-gold (Ag-Au) alloy nanohole arrays (alpha NHA) for ultra-sensitive plasmonic label-free detection of Escherichia Coli (E. coli). Large-area alpha-NHA were fabricated by using nanoimprint lithography and concurrent thermal evaporation of Ag and Au. The completely miscible Ag-Au alloy exhibits an entirely different dielectric function in the near infra-red wavelength range compared to mono-metallic Ag or Au. The alpha-NHA demonstrate substantially enhanced refractive index sensitivity of 387 nm/RIU, surpassing those of Ag or Au mono-metallic nanohole arrays by approximately 40%. Moreover, the alpha-NHA provide highly durable material stability to corrosion and oxidation during over one-month observation. The ultra-sensitive alpha-NHA allow the label-free detection of E. coil in various concentration levels ranging from 10(3) to 10(8) cfu/ml with a calculated limit of detection of 59 cfu/ml. This novel alloy plasmonic material provides a new outlook for widely applicable biosensing and blo-medical applications.