We previously reported the development of direct X-ray fluorescence (XRF) imaging system for quantitative imaging of gold nanoparticles (GNPs) on a benchtop setting. This system is implemented with an ordinary polychromatic X-ray source and, by detecting gold L-shell XRF photons, is capable of detecting/imaging trace amounts of gold nanoparticles (GNPs), on the order of parts-per-million (ppm), under the calibration conditions. For routine XRF imaging of biological samples (e.g., explanted tumors) containing ppm-level GNPs, this system needs to be tested further under more realistic imaging conditions. Thus, we developed a GNP-loaded tissue-mimicking phantom and performed a phantom imaging study using our benchtop XRF imaging system. For the tissue-mimicking phantom construction, the GNP-filled capillary tubes with known GNP concentrations were placed in an 'E' shaped pattern and sandwiched between three layers of cheese. The results (i.e., XRF map) from the scanning of this phantom showed that the 'E' shape of the phantom was well visible in the XRF map and all three arms and the stem of 'E' were spatially resolved within ∼2 mm. The measured GNP concentrations were in good agreement with the known GNP concentrations.