Pyocyanin is a virulence factor solely produced by the pathogen Pseudomonas aeruginosa. Pyocyanin is also a redox active molecule that can be directly detected by electrochemical sensing. A nanograss (NG) based sensor for sensitive quantification of pyocyanin in sputum samples from cystic fibrosis (CF) patients is presented here. The NG sensors were custom made in a cleanroom environment by etching nanograss topography on the electrode surface followed by depositing 200 nm gold. The NG sensors were utilized for amperometric quantification of pyocyanin in spiked hypertonic saline samples, resulting in a linear calibration curve with a R2 value of 0.9901 and a limit of detection of 172 nM. The NG sensors were applied in a small pilot test on five airway samples from five CF patients. The NG sensor was capable of identifying P. aeruginosa in the airway samples in 60 s without any sample pretreatment.