Using Surface enhanced ROA (SEROA), novel results are achieved by combining Raman Optical Activity (ROA) and resonance Surface Enhanced Raman Scattering (SERRS), applied on myoglobin. The novelty of this work is ascribed the first time reporting on chiral results of a study performed on a protein at single molecule level. The results of this work, using silver nanoparticles and a laser excitation of 532 nm, became only feasible when the concentrations of nanoparticles, aggregation agent NaCl and the studied molecule were optimized in a series of systematic optimization steps. The spectral analysis has shown that the SERS effect behaves consequently, depending on the concentration ratio of each component, i.e., myoglobin, Ag colloids and NaCl. Accordingly, it is shown here that SERS intensity has its maximum at certain concentration of these components, whereas below or above this value, the intensity decreases. The optimization results can be considered as a completion of the hitherto known phenomenon "dilution effect", which takes only account for higher concentrations. Furthermore, the optimization of the parameters seems to be necessary for a successful SEROA measurement, which enables chiral study of a protein on the single molecule level, in which the concentration and acquisition time are no longer an impediment.