Purpose: The objective of this study was to develop a fatty acid database (DB) for estimation of intake levels of fatty acids in the Korean population, using data from the Korea National Health and Nutrition Survey (KNHANES). Methods: Analytical values of fatty acids in foods were collected from food composition tables of national institutions (National Fisheries Research & Development Institute, Rural Development Administration), Japan Ministry of Education, Culture, Sports, Science and Technology, US Department of Agriculture, and journal articles that previously reported analytical fatty acid content of some Korean foods. The coverage of fatty acids was C14:0, C16:0, C18:0, C18:1, C18:2 n-6, C18:3 n-3, C20:5 n-3 (EPA), C22:6 n-3 (DHA), SFA, MUFA, and PUFA (n-3, n-6, n-9). The fatty acids DB covered a total of 5,144 food items used in the KNHANES nutrition survey. The food items were preferentially filled with analytical values of the collected data source. An analytical value for each food item was selected based on the priority criteria and the quality evaluation of data sources. Missing values were replaced with calculated or imputed values using the analytical values of similar food items from the data source. Results: A total of 1,545 analytical values, 2,589 calculated values, and 1,010 imputed values were included in the fatty acid DB. The developed fatty acid DB was applied to 2,112 food items available for 2011 KNHANES data. Mean intake levels of total fatty acids and saturated fatty acids were 40.3 g/day and 13.2 g/day, respectively. The estimation of total fatty acid intake was 84.3% (men 83.2%, women 86.0%) of daily total fat intake. Conclusion: This newly developed fatty acid DB would be helpful in determining the association of fatty acids intake and related health concerns in the Korean population.