An ultrasonic extraction-multiskan spectrum-microtitration assay method has been developed to detect the total petroleum hydrocarbons in environmental matrix. With a study into the parameters and variables that affected the extraction and detection of petroleum, it was found that the optimal conditions for extraction and detection are as follows: the detecting wavelength being 304 nm, using petroleum ether as extracting reagent, soil and liquid ratio being 1∶4, ultrasonic extracting two times with each one lasting 20 minutes, and ultrasonic extracting power, 100 W. Under these conditions, the recovery rates of total petroleum hydrocarbons ranged from 88.4% to 101.6%, with all the relative standard deviations less than 4.7%. Compared to the traditional extracting method which involves the use of lots of organic reagents such as dichloromethane, it would be more environment-friendly, for it is less harmful to the environment. Replacing ultraviolet spectrophotometer with multiskan spectrum, the samples can be detected more easily and rapidly with a relatively small amount of solution. Consequently, this method proves to be an efficient, green, rapid and inexpensive approach for extraction and determination of total petroleum hydrocarbons from environmental samples, which can be widely used in the quantitative determination of the trace environmental samples of petroleum contamination.