The rapid detection of bacterial metabolites responsible for cell communication is important for the early diagnosis of bacterial infection and treatment with appropriate antibiotics. Surface-enhanced Raman spectroscopy (SERS) has provided amplified molecular fingerprints, serving as one of the most promising nondestructive and label-free direct detection methods. However, direct analysis of complex bacterial cultures using the conventional SERS approach is difficult. Here, we design a new paper-based 3D SERS substrate and establish a facile one-pot Au electrodeposition protocol to directly detect pyocyanin, a signaling molecule of Pseudomonas aeruginosa, secreted in the culture medium, without pretreatment steps. During one-pot Au electrodeposition in the presence of an Au precursor and culture medium, the bacterial pathogen molecule (i.e., pyocyanin) is captured in the interstitial voids of the 3D hydrogel matrix among growing Au grains, resulting in high amplification of the Raman signal intensity. Because the hydrogel skin excludes macromolecules (i.e., proteins) in the culture medium, thereby maintaining an uncontaminated surface, our approach achieves the direct and rapid detection of pyocyanin in a complex bacteria culture sample without any purification steps. Therefore, the pyocyanin concentration during bacterial culturing is easily and directly monitored, which is important for the early diagnosis of infection and in-depth studies of bacterial cell communication.