With the increase of human activities in industry and agriculture, the species and the toxic effect of water pollutant are getting more and more complex. Therefore, the online monitor of the aquatic toxicity has been imminent. Advantaged in rapidness, sensitivity and cheapness, the luminescent bacteria method can be applied into the online monitor, which can objectively reflect the toxicity of toxic substances as well as the change of it. Photobacterium phosphoreum is used as the test strains in this method, and the standard is 10mg/L 7H2O·ZnSO4, the best test temperature is 15℃ and the strain recovery time stabilization time after recovery is 24hours. The proposed method was successfully applied to measure actual water sample, proforma rate of addition standard is between 95.94% and 97.02%.