In this study, the AuNP k-shell fluorescence spectra were measured by using a 100 mm long collimator to improve the detectability. The AuNPs were suspended in deionized water at different concentrations. The AuNP suspensions were excited by a micro focal spot x-ray tube with 130 kVp, 300 μA x-ray exposures. The emissions of the AuNP fluorescence were measured by a spectrometer located with an angle of 90 degrees with respect to the excitation beam. The fluorescence acquisition durations for each concentration mode were 3000 s. A 1.0 mm Pb filter and a 1 mm Al filter were utilized to optimize excitation beam and fluorescence emission, respectively. As a result, the k-shell fluorescence peaks, 66.99 keV and 68.80 keV of AuNP, were measured and observed in 0.1, 0.2, 0.4, 0.8, 1.0, 2.0, 4.0 mg/mL concentration modes. The linear relationship between the AuNP suspension concentrations and the number of photons of the fluorescence peaks were observed in the range of 0.1–4.0 mg/mL. The results of experimental measurements demonstrated up to 0.1 mg/mL (0.01 % in weight concentration) detectability.