We have explored the effects of the experimental parameters on the surface-enhanced Raman scattering (SERS) intensities of NO3− and proteins observed by a heat-induced SERS method developed by our group. The results have shown that a strong SERS signal can be obtained at pH 4.0, using an Ag colloid prepared with the reduction time of 15 min (the average size of Ag nanoparticle is 56.5 nm) dilution prepared Ag colloid by a factor of 2 by use of a 5 mM citrate buffer, using 6 mM NaNO3 and drying the sample at 100 °C, respectively. Based on the results, two possible mechanisms for proteins to form SERS hot sites during the sample preparations are proposed. A semi-quantitative SERS detection of ribonuclease B has been investigated. Also, NaNO2, Mg (NO3)2, MgSO4 and Na2SO4 have been found to be suitable for the heat-induced SERS method. Importantly, samples prepared by the heat-induced SERS method are so stable that these samples can be used as a standard and transferred to different laboratories for direct comparison. Namely, it can overcome uncontrollable aggregation of Ag colloids in a solution sample. All these advantages and the simplicity of experimental setup have demonstrated that the heat-induced SERS method using NaNO3 as an electrolyte is very promising for label-free routine and quantitative detection of proteins. Copyright © 2011 John Wiley & Sons, Ltd.