Protein detection using surface-enhanced Raman scattering (SERS) usually requires electrolytes to yield an enhanced SERS signal. However, the adsorption mechanism of electrolyte and protein to Ag colloid is not yet clearly understood. In this work, we have investigated co-adsorption of NO 3- and lysozyme to Ag colloid using SERS. Three experimental factors including concentration of lysozyme (10-5 and 10-6 M), concentration of NO3- (0, 1, 2, 3 and 5 mM) and drying temperature (25 and 100 °C) have been studied. The results have shown that the co-adsorption of the adsorbates (lysozyme and NO 3-) on a SERS substrate and the non-absorption of NO 3- on the substrate can be controlled by using different experimental conditions. The co-adsorption manner of lysozyme and NO 3- is consistent with the mechanism of double-layer adsorbates when a protein adsorbs on a solid/liquid interface. The variation in protein conformation, especially the main-chain conformation, seems to affect the adsorption manner of the adsorbates. It has been found that the final adsorption result is not affected by the addition sequence of lysozyme and NO3- during the sample preparation.