X-ray luminescence computed tomography (XLCT) is an emerging hybrid molecular imaging modality. In XLCT, high energy x-ray photons excite phosphors emitting optical photons for tomographic image reconstruction. During XLCT, the optical signal obtained is thought to only originate from the embedded phosphor particles. However, numerous studies have reported other sources of optical photons such as in air, water, and tissue that are generated from ionization. These sources of optical photons will provide background noise and will limit the molecular sensitivity of XLCT imaging. In this study, using a water-cooled electron multiplying charge-coupled device (EMCCD) camera, we performed luminescence imaging of water, air, and several tissue mimicking phantoms including one embedded with a target containing 0.01 mg/mL of europium-doped gadolinium oxysulfide (GOS:Eu3+) particles during x-ray irradiation using a focused x-ray beam with energy less than the Cerenkov radiation threshold. In addition, a spectrograph was used to measure the x-ray luminescence spectrum. The phantom embedded with the GOS:Eu3+ target displayed the greatest luminescence intensity, followed by the tissue phantom, and finally the water phantom. Our results indicate that the x-ray luminescence intensity from a background phantom is equivalent to a GOS:Eu3+ concentration of 0.8 μg/mL. We also found a 3-fold difference in the radioluminescence intensity between liquid water and air. From the measurements of the emission spectra, we found that water produced a broad spectrum and that a tissue-mimicking phantom made from Intralipid had a different x-ray emission spectrum than one made with TiO2 and India ink. The measured spectra suggest that it is better to use Intralipid instead if TiO2 as optical scatterer for future XLCT imaging.