This paper describes a gas chromatography-mass spectrometric method of l-hydroxypyrene (1-HOP) and 2-naphthol (2-NAP) in fish and shellfish. Alkali hydrolysis method in this study was chosen and optimized to the reaction condition for 90 min at 90°C in a 2.0M KOH solution. For five independent determinations at 0.2 and 1.0 μg/kg, the coefficient of variation was less than 5.1%. This method was used to assess the long-term influence of spilled crude oil on marine ecosystems and analyze fifty-two shellfish samples taken in the near of the accident region of the Hebei Spirit oil spill. 2-NAP and 1-HOP were detected in the mean concentration range of 0.09-12.42 and 0.03-0.06 μg/kg, respectively. 2-NAP was detected in a high concentration range in shellfishes gathered in 2 months after the accident and it decreased rapidly to 6 months after that. The results showed that 2-NAP might be an important biomarker in biota contaminated with crude oil.