This paper discusses a method for assembling Au-Ag alloy nanoparticles (NPs) using direct current (DC) electric field to fabricate highly active SERS substrates. Different nanostructures could be obtained by regulating the intensity and action time of DC electric field. Under the condition of 5mA*10 min, we obtained Au-Ag alloy nano-reticulation (ANR) substrate with excellent SERS activity (Enhancement factor on order of magnitude of 106). ANR substrate has excellent SERS performance due to the resonance matching between its LSPR mode and excitation wavelength. The uniformity of the Raman signal on ANR is greatly improved than bare ITO glass. ANR substrate also has the ability to detect multiple molecules: ANR substrate can respectively detect Rh6G and CV molecules with a concentration as low as 10-10 M and 10-9 M and the Raman spectral intensity of the probe molecules on the surface of the ANR substrate has good linear correlation with the molecular concentration (R2 > 0.95). In addition, ANR substrate can detect both thiram and aspartame (APM) molecules far below (thiram for 0.0024 ppm and APM for 0.0625 g/L) the safety standard, which demonstrate its practical application potential.