Abstract: The surface hydrophobicity of microorganisms is one of the main factors affecting the absorption and degradation of hydrophobic organic matter. Improving the surface hydrophobicity of microorganisms can help them adhere to the surface of petroleum hydrocarbon, thus effectively reducing the mass transfer distance of petroleum hydrocarbon and improving its bioavailability. A strain with surface hydrophobicity that can use petroleum hydrocarbon as the only carbon source was isolated through enrichment culture from the polluted seawater of Dinghai Port in Zhoushan City. The physiological and biochemical identification and 16S rRNA gene sequence evolution analysis of the strain were conducted. The effects of incubation time, salinity, substrate and surfactants on the surface hydrophobicity of this strain were studied. The results showed that this strain belonged to the genus Pseudomonas, which was the closest relative to Pseudomonas paralactis, and was named as strain H2. The surface hydrophobicity increased with the increase of culture time when the strain was in logarithmic growth and stable stages. In the aging stage, the surface hydrophobicity decreased gradually with the increase of culture time. The increase of medium salinity reduced the surface hydrophobicity of the strain. When the hydrophobic diesel oil was used as the only carbon source, the surface hydrophobicity of the strain was much higher than that of hydrophilic glucose. The surface hydrophobicity of strain H2 was changed by adding surfactants in the culture process. The surface hydrophobicity was improved when non-ionic surfactants of Tween20 and Tween80 were added at the mass concentration of 30 mg/L and 14 mg/L, respectively. However, the anionic surfactant (rhamnolipid) and ampholytic surfactant (lecithin) reduced the surface hydrophobicity of strain H2.