Traditional high-throughput methods for identification of chemicals with liver tumor promotion potentials are based on established cancer cell lines, and rapid and cost-effective high-throughput screening assays in whole organisms are presently lacking. In this study, a transgenic zebrafish liver cancer model was employed to develop a method that could be used to identify chemicals with liver tumor promotion effect quickly and accurately. The method consisted of three parts, including exposure preparation, exposure process and image acquisition. In brief, after chemical exposure for 7 days, 96-well plate exposure system for zebrafish larvae was assessed by microplate reader. Then, the liver cancer promoting potential chemicals were evaluated by field area and field average intensity of fluorescence. The results were further validated by conducting histopathological examination. Our data demonstrated that the high-throughput screening assay developed in this study was reproducible and could be used to rapidly screen chemicals with liver tumor promoting potentials by using tris-(2-chloropropyl)-phosphate (TDCIPP) as a positive control. Furthermore, some other positive chemicals found in previous studies and environmental compounds were assessed using the established method. Results indicated that 86.7% of the positive chemicals and five environmental compounds out of seventeen compounds could enhance liver tumor progression.