The changes of wastewater toxicity to activated sludge along the treatment processes from water intake to the aerobic biological unit in an integrated petrochemical wastewater treatment plant were investigated. The assay was done based on three toxicity characterization methods: oxygen uptake rate of activated sludge, dehydrogenase activity of activated sludge and acute toxicity to luminescent bacteria. Moreover, the three-dimensional fluorescence spectra of effluents from each process unit were analyzed and the relationship between fluorescence characteristics and the toxicity profiles was revealed. The results showed that the influent petrochemical wastewater had an inhibition on the oxygen uptake rate of nitrifier up to 50%~60%. After the treatment processes prior to the aerobic biological treatment unit, the inhibition of the treated wastewater on activated sludge still reached 30%. Among them, hydrolysis acidification and anoxic treatment were found to decrease the wastewater toxicity obviously. Based on the comprehensive comparison of the results from these three methods, the oxygen uptake rate method is more suitable for the toxicity evaluation of petrochemical wastewater on activated sludge. As to the fluorescence spectra of the water samples, all the fluorescence peaks appeared in the region of λ_ex/λ_em=200-300/250~400nm, and the fluorescence intensity of peak C (λ_ex/λ_em=225/340), peak E (λ_ex/λ_em=275/325) and peak F (λ_ex/λ_em=275/335) were positively correlated with the toxicity at a certain extent. The fluorescence peaks with emission wavelength of 325~340nm are recommended to be used for rapid and simple monitoring the toxicity of petrochemical wastewater to activated sludge.