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There are a number of bioactive compounds in freshwater fish brains, and their functional roles have not been clearly elucidated. NMR-based metabolic profiling could enable rapid characterization of the nutritional composition a fish's brain. Here, two kinds of freshwater fish brains were investigated, crucian carp and yellow catfish. A 1H-NMR based metabolomic approach was used to illustrate the nutritional components of these two kinds of brain. At first, the microwave method was utilized to cease the activity of the enzymes in the brain, and the chemicals were extracted for NMR analysis. These two kinds of brain had significant differences in metabolic patterns, and the chemical compositions of the yellow catfish brain were similar to those of rodent and human brains. Furthermore, most of the different metabolites were significantly higher in the yellow catfish, except for acetamide. This study could provide comprehensive information regarding the utilization of fish heads during processing of fish and dietary nutrition guidance.