The establishment of marine protected areas has been wildly recognized as an important way to conserve the biological diversity and to prevent deterioration of marine eco-environment. The sound implementation of eco-compensation is a critical measure to guarantee the effective construction and management of marine protected areas and promote the equalized opportunity of regional economic development. How to determine eco-compensation standard is one of difficult issues in eco-compensation research, and also a core aspect of ecological civilization construction in China. Opportunity cost method has been widely utilized in some practical fields such as grassland eco-compensation, watershed eco-compensation, agricultural eco-compensation, and eco-compensation in nature reserves. By comparison, it is found to be lag behind inresearch and case studies of the eco-compensation standard of marine protected areas. From the view of opportunity cost of eco-compensation, we used the cardinal data of opportunity cost eco-compensation, regional adjustment coefficient, zonal compensation coefficient, and development limit compensation coefficient to develop an approach of estimating eco-compensation standards for ecological conservation of marine protected areas, and then carried out empirical analysis in 14 national marine protected areas in 8 provinces or municipalities of China. The results showed that the estimated eco-compensation amounts were 0.66×10⁴ to 10.69×10⁴ RMB/km² in those case areas. Among them, the eco-compensation amounts of the Changli Golden Coastal National Nature Reserve of Hebei and Tianjin Ancient Coast and Wetland National Nature Reservewere significantly higher than those of other protected areas with 10.69×10⁴ RMB/km² and 10.47×10⁴ RMB/km², respectively. In contrast, the average eco-compensation amount was 1.70×10⁴ RMB/km² for the left 12 ones.