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