Based on the simulative exposure experiments of nitrobenzene to two small experimental fishes, Medaka (Oryzias latipes) and Chinese Rare Minnow (Gobiocypris rarus), it was found that nitrobenzene could decrease in a linear way in the static aquatic system. Both fishes could accumulate dose-related levels of nitrobenzene quickly and eliminate the compound rapidly when they were transferred to clean water. The Chinese rare minnow showed more sensitivity to the acute toxicity exposure of nitrobenzene than Medaka. Typical molecular biomarkers in oxidative defense system including superoxide dismutase (SOD), catalase (CAT) showed the chemical induced alterations. The changes of acetylcholinesterase (AChE) activities indicated that nitrobenzene might affect the normal neural function. Nitrobenzene exposure could also lead to obvious damaging effects on the target organs, such as gill and liver by hitopathological studies.