Although polycyclic aromatic hydrocarbons (PAHs) are considered as toxic and refractory pollutants, their biodegradation can be facilitated by biosurfactants. However, few studies have been performed to understand the potential isolation and application of biosurfactant-producing microorganism for promoting the in-situ removal of PAHs from wastewaters. In this work, a biosurfactant-producing strain S5 isolated from coking wastewater was identified as Pseudomonas aeruginosa. The biosurfactant produced by strain S5 was determined as glycolipid with a critical micelle concentration (CMC) of 96.5 mg/L, and reduced the surface tension from 72.2 to 29.6 mN/m. Addition indigenous P. aeruginosa S5 to coking wastewater effectively promoted the biodegradation of high weight molecular (HWM) PAHs (reduction from 9141.02 to 5117.16 mu g/L in 15 days) in sludge phase. The results showed that the removal of PAHs in the sludge was enhanced by inoculating indigenous biosurfactant-producing microorganism in coking wastewater serving as an in-site remediation technology.