Early stage detection of Pseudomonas infections is life-saving, especially in the case of patients with cystic fibrosis. Pyocyanin (PYO) is a specific metabolite of the Pseudomonas aeruginosa bacteria, and detection of it directly in the sputum can significantly reduce the diagnosis time of the infection. In the present study, aiming to achieve this goal, simple and cost-effective surface-enhanced Raman spectroscopy (SERS) detection platform was proposed. For this, silicon nanowires (SiNWs) matrix, produced by metal-assisted chemical etching of silicon substrates was variously modified by noble metal (silver (Ag) and gold (Au)) nanoparticles (NPs) and tested for PYO detection in complex matrix of artificial sputum. SERS measurements revealed that for the PYO detection is beneficial to have Ag NPs on the bottom of SiNWs and deposited bimetallic Ag/Au NPs on the top. Such substrate showed good point-to-point and batch-to-batch signal reproducibility and could detect PYO in artificial sputum down to 6.25 µM concentration, which is the required sensitivity for clinical applications.