Positively charged nanoparticles to be used as substrates for surface-enhanced Raman scattering (SERS) were prepared by coating citrate-reduced silver nanoparticles with the cationic polymer poly-l-lysine. The average diameter of the coated nanoparticles is 75 nm, and their zeta potential is +62.3 ± 1.7 mV. UV-vis spectrophotometry and dynamic light scattering measurements show that no aggregation occurs during the coating process. As an example of their application, the so-obtained positively charged coated particles were employed to detect nanomolar concentrations of the anionic chromophore bilirubin using SERS. Because of their opposite charge, bilirubin molecules interact with the coated nanoparticles, allowing SERS detection. The SERS intensity increases linearly with concentration in a range from 10 to 200 nM, allowing quantitative analysis of bilirubin aqueous solutions.