Fish oil is a popular nutritional product consumed in China. The beneficial effects of fish oil have been attributed to docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA). Hence, it is crucial to develop a rapid and precise method to determine the contents of EPA and DHA in fish oil. In this study, a rapid and accurate proton nuclear magnetic resonance method for the quantification of DHA and EPA was developed. Dimethyl terephthalate was selected as an internal standard, and the signals at 2.391 ppm for DHA and at 1.697 ppm for EPA were chosen for the quantification. Validation of the method was performed in terms of specificity, precision, and stability. The results indicated that the method was precise and in line with the China Food and Drug Administration guidance. The method has been successfully applied to characterize fish oil capsules obtained from four pharmaceutical companies. This study indicated that the rapid 1H NMR method is suitable for the quality control of fish oil.