Chemotaxis of pathogen toward the mucus layer of fish plays an important role in its colonization in the mucus. Chemotaxis is considered to be one of the virulent mechanisms of pathogenic bacterium. For a better understanding of the chemotactic response of pathogenic Vibrio fluvialis toward the skin mucus of Paralichthys olivaceus so that preventive measures can be devised to reduce the bacterial colonization, the influences of bacteria concentration, incubation time, and environmental factors, such as temperature, pH, salinity and carbohydrate, on the bacterial chemotaxis were investigated by the method of modified capillary assay and isotope tracer. The results showed that the bacteria number of chemotactic V. fluvialis toward skin mucus of P. olivaceus increased with the bacteria concentration and incubation time, respectively. Saturation of bacterial chemotaxis was obtained after incubating at room temperature for 60 min; the chemotactic bacteria increased with the incubation temperature from 5 ℃ to 15 ℃ and peaked at 15 ℃, and optimal chemotaxis was observed at pH 8; the bacterial chemotaxis weakened with the increase of NaCl concentration from 0.8 % to 3.6 %. Among 8 kinds of carbohydrate tested, mannitol, lactose and seminose promoted the bacterial chemotaxis remarkably. The results indicate that pathogenic V. fluvialis has a strong chemotaxis toward the skin mucus of P. olivaceus, and this chemotaxis is influenced remarkably by environmental factors, such as temperature, pH value, salinity and carbohydrate. The chemotaxis characteristics revealed in this paper would be helpful for the design of effective prophylactic strategies for P. olivaceus’ culture.