Marine oil spill affects the marine flora and fauna to a greater extent in the marine ecosystem. Many marine bacteria have the ability to degrade petroleum hydrocarbons by producing biosurfactants and alkane hydroxylase enzymes. Biosurfactant enhances bioavailability of crude oil increasing the dispersion of hydrocarbons in the water by expanding the contact surface area due to emulsification. In this study, we have isolated and characterized crude oil-degrading Pseudomonas aeruginosa from oil-polluted Mumbai harbor water. The biosurfactant-producing ability of the strain MR21 was tested by different biosurfactant screening methods like the blue agar method, hemolysis, drop collapse assay, oil displacement method, surface tension reduction, and emulsification index. The strain MR21 showed a positive response for all these methods. Biochemical characterization indicated that this strain is able to hydrolyze casein and Tween 80 by producing proteases and lipases. The gas chromatography analysis indicated complete degradation of crude oil by strain MR21 after 30 days. The catabolic genes responsible for alkane degradation such as alkane hydroxylase gene (alkB) and flavin-binding monooxygenase gene (almA) were detected in this strain MR21 by polymerase chain reaction. This strain could be useful for bioremediation of marine oil spills in the environment.