The bioluminescent platform based on a genetically engineered luciferase NanoLuc and its synthetic substrate furimazine is widely used in modern molecular biology for optical imaging and therapy of deep tissue tumors. Taking into account the perspectives of using this system in molecular biology research, a detailed study of the toxicity of furimazine in vitro and in vivo is of great interest. In this work, the cytotoxicity of furimazine in vitro was studied using four different cell lines. Systemic toxicity of furimazine in vivo was investigated under conditions of prolonged administration of the substrate in animals. It has been found that seven-day intravenous injection of the substrate in animals causes hydropic dystrophy of liver and necrosis of hepatocytes. Splitting of the dose into several injections reduces hepatotoxicity of furimazine.