Gastric cancer is the second leading cause of cancer mortality worldwide. Understanding the multistep process of carcinogenesis of gastric cancer is pivotal to develop novel therapeutic strategies. Molecular imaging in preclinical cancer models bridges the gap of laboratory-based experiment and clinical translation. To this end, the human gastric cancer cell line SGC-7901 was established to stably express luciferase and GFP by lentiviral transduction (SGC7901-Luc-GFP). Preclinical models were developed by orthotopic transplantation of SGC-7901-Luc-GFP into the sub-serosal layer of the stomach of immunocompromised mice. Tumor progression and therapeutic responses were dynamically tracked by bioluminescence imaging (BLI). Bioluminescence tomography (BLT) was used to monitor stereoscopic morphological and signal changes during tumor progression. Good correlation between cell number and bio-luminescence/fluorescence intensity was observed (R(2)=0.9983/r(2)=0.9974) in vitro. Tumor progression and therapeutic response could be successfully followed directly by BLI. Importantly, BLT provided a more accurate spatial location and tomographic quantification of the internal lesion. In conclusion, our novel bioluminescence-based preclinical gastric cancer models enable superior, noninvasive monitoring gastric cancer progression and their drug responses. The BLT technique in particular, may have great potential for future oncological studies.