In this study, we use near-infrared spectroscopy (NIR) detection technique to establish the database for differentiation of frozen-thawed fish products. The method is based on the release of the β-hydroxyacylCoA-dehydrogenase (HADH) from mitochondria during freezing. The relationship between the HADH activity of fish products and the NIR database could provide the information for us to separate fresh and frozen-thawed fish products. At the same time, combined with different freshness indicators, and measured freshness.