An inexpensive, simple, highly sensitive, and rapid fluorimetric method was developed for the analysis of pseudoephedrine hydrochloride at trace levels. The method is based on the recovery of fluorescence of Rh6G dye due to the interaction of pseudoephedrine hydrochloride with Rh6G-Au NPs complex, which results in the release of Rh6G from the complex and halting fluorescence resonance energy transfer between Rh6G and Au NPs. The intensity of fluorescence was directly proportional to the concentration of the analyte, which was used for its determination. Experimental factors were optimized by response-surface methodology. Under optimum conditions, the calibration curve was linear over the range of 15-150 ng mL −1 and the limit of detection (LOD) was 10 ng mL −1 . Percent relative standard deviation (n = 5) for determination of 50 ng mL −1 pseudoephedrine hydrochloride was 3.74%. The method was successfully used for determining the analyte in human blood serum and in pharmaceutical formulations. The possible mechanistic description of the analytical reaction was proposed on the basis of TEM, FT-IR, and fluorescence spectra analysis.