A large-scale-integration-based multiple amperometric biosensor (Bio-LSI) was used to obtain images of oxygen concentration around zebrafish embryos and monitor their respiration activity after valinomycin at different concentrations was injected. The regions with low reduction current of oxygen coincided with the embryo positions. The reduction current decreased immediately after adding the valinomycin owing to the increase in the respiration activity of the embryos. The decrement of the reduction current increased with increasing valinomycin concentration. The estimation system is very simple because no alignment is required between the embryos and electrodes arrayed on the bottom of the chamber. In general, the use of zebrafish embryos for toxicity tests has been expected as an alternative for animal tests. However, a relatively long time is required to estimate the mortality and morphological changes of the embryos to chemical substances by a standardized method. The use of the present system is expected to allow the rapid and simple sensing of the effect of chemical substances on the respiration activity of zebrafish embryos.