We analyze microstructured multilayer films of poly(ethyleneimine) (PEI) and DNA by employing Raman and surface enhanced Raman spectroscopy (SERS). The microstructuring of the samples allows a simultaneous measurement of signal and reference in a single analytic process. Silver nanoparticles are implemented in the microstructured multilayers for SERS measurements. The recorded SERS spectra of PEI/DNA are dominated by the Raman bands of the DNA bases which show a larger mean enhancement than bands belonging to DNA backbone vibrations. Our results show that the combination of SERS and microstructured multilayer films provides an adapted way to characterize the polyelectrolytes as well as to measure the enhancement factor and the distance dependence for the SERS active silver nanoparticles. Furthermore, microstructured polyelectrolyte films containing SERS active nanoparticles are used for sensing molecules.