Abstract: In recent years, ichthyotoxic algal bloom has brought huge marine culture economy losses to China. Reactive oxygen species and hemolytic toxins produced by ichthyotoxic algae were regarded as major factors of fish killed. To understand the toxic mechanism and mode of action of ichthytoxic algae on fish, the effect of hydrogen peroxide (substituted for reactive oxygen species) and digitonin (substituted for hemolytic toxins) on Oryzias melastigma were investigated by single and combined experiments. The results showed that the LC₅₀ of digitonin on O.melastigma within 0.5 h and 1 h were 3.44 and 1.25 μg/mL, respectively. The LC₅₀ of hydrogen peroxide on O.melastigma was 144.5 mmol/L within 1 h. By using the analysis methods of additional index, toxic unit, similarity parameter and mixture toxic index, a synergistic effect of hydrogen peroxide and digitonin on O.melastigma was found. Fish unit (FU) was established according to the digitonin experiment results, which provided a method of evaluating toxicity of ichthyotoxic algae. One FU was the exposure concentration of digitonin killed an O.melastigma fish weighed 20 mg and 60 days old within 0.5 h. It's equal to the digitonin concentration of 3.44 μg/mL.