Purpose: To develop a high sensitivity L‐shell x‐ray fluorescence imaging system that locates and quantifies sparse concentrations of gold nanoparticles (GNPs) using a polychromatic benchtop x‐ray source. Methods: An L‐shell x‐ray fluorescence imaging system was built with a benchtop polychromatic x‐ray source and a Si‐PIN detector. Water‐filled cylindrical tubes (12 mm in diameter) loaded with GNPs at 2%, 1%, 0.5%, 0.05%, and 0.005% GNP by weight were served as calibration phantoms. An imaging phantom was created using the same cylindrical tube but filled with water‐equivalent gel containing structures mimicking GNP‐loaded blood vessel and 1 cc tumor. The phantoms were irradiated by a 3‐mm diameter 65 kVp x‐rays filtered by 1 mm aluminum. Fluorescence/scatter photons from the phantoms were detected at 90° with respect to the beam direction using a Si‐PIN detector with a pin‐hole collimation. For the imaging phantom, the detector was translated horizontally and vertically in 0.3‐mm steps to create a 2‐D fluorescence image of the phantom. The net L‐shell fluorescence signal from GNPs was extracted from background, and then corrected for detector efficiency and in‐phantom attenuation using a fluorescence‐to‐scatter normalization algorithm. The corrected signal from the calibration phantoms was used to create a calibration curve showing a linear relationship between corrected fluorescence signal and GNP mass per image voxel. Results: The results suggest that the current setup can detect a GNP mass of 500 ng (or 0.5 ppm) contained within each image voxel (0.0173 cc). The 2‐D fluorescence image properly correlated the known spatial distribution and amount of GNPs within the imaging phantom. Conclusion: L‐shell fluorescence imaging can be a highly sensitive tool that has the capability of simultaneously imaging the spatial distribution and determining the local concentration of GNPs presented within ex‐vivo samples and superficial tumors during pre‐clinical small animal studies. Supported by NIH/NCI grant R01CA155446; NIH/NCI grant R01CA155446.