Sissoo spinach, also known as Alternanthera sissoo or A. sissoo, is commonly found in Brazil and Malaysia. It is a popular plant among Malaysians due to its ability to thrive in various environmental conditions and weather. Additionally, it is believed to possess therapeutic properties and is considered a good source of vitamins and minerals with beneficial results. This study is aimed to evaluate the toxicity level of A. sissoo using zebrafish. The plant was extracted by ethanol extraction and two-fold serial dilution was performed to prepare 13 series of different concentrations. The zebrafish embryo’s survival rate decreased with increasing concentration, and no survival was observed at concentrations above 125 µg/ml to 1000 µg/ml. On the other hand, hatchability increased as the concentration of A. sissoo decreased, with no hatching observed at concentrations above 125 µg/ml to 1000 µg/ml. Zebrafish heartbeat remained within normal range at concentrations ranging from 0.244 µg/ml to 62.5 µg/ml. Scoliosis was not observed in zebrafish embryos at A. sissoo ethanol extract concentrations ranging from 0.244 µg/ml to 62.5 µg/ml. At concentrations of 0.244 µg/ml to 62.5 µg/ml, melanin pigment was present in the embryo. The LC₅₀ value obtained from ethanol extraction of A. sissoo was 40.343 µg/ml, indicating that it is safe for consumption at appropriate concentrations. However, the consumption of high concentrations of this plant extract may result in mild toxicity.