Zebrafish are widely used for detecting toxic agents because of their unique advantages. The conventional zebrafish-based tests use lethal rates and morphological changes as criteria to evaluate the toxicity. To increase the sensitivity of using zebrafish to detect toxic agents, a fluorescence resonance energy transfer-based apoptotic biosensor was introduced into zebrafish genome to generate transgenic sensor zebrafish. Seven chemicals including heavy metals, nanomaterials and DNA-damaging agents were used to treat the sensor zebrafish to determine the sensitivity of the sensor zebrafish. The results showed that sensor zebrafish can detect the toxicity of the tested agents with single-cell sensitivity. Using the sensor zebrafish, we found that, at 100 nM, heavy metal cadmium (Cd) induced apoptosis of zebrafish cells, while no obvious morphological or behavioral changes were observed from the sensor zebrafish. Even at 44.5 nM (the maximum allowable concentration in drinking water), Cd induced a significant increase of apoptosis in sensor zebrafish. ZnO nanoparticles caused apoptosis in sensor zebrafish at a very low concentration of 100 ng/mL. DNA-damaging agents induced the apoptosis of many cells in sensor zebrafish. The sensor zebrafish are much more sensitive than the conventional zebrafish-based tests and can serve as a powerful tool for detecting toxic agents.