Abstract: To investigate the flavor changes of pre-cooked crayfish after frozen storage and reheating, flavor analysis was performed using an electronic nose (E-nose) combined with headspace-solid phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS). The key flavor substances were then subjected to orthogonal partial least squares-discriminant analysis (OPLS-DA) and relative odor activity value (ROAV) for in-depth exploration. The highest sensory evaluation score was observed in crayfish pre-cooked for 4 min. After frozen storage and reheating, the Y4-D30-F2 group (pre-cooked for 4 min, frozen for 30 days, reheated for 2 min) achieved the highest sensory score. Significantly higher response values were recorded by the E-nose on sensors W1W, W1S, and W2W compared to other sensors, indicating elevated levels of inorganic sulfides, alkanes, aromatic compounds, and organic sulfides in pre-cooked crayfish. GC-MS analysis identified 39 types of volatile flavor compounds in the pre-cooked crayfish, which increased to 75 after frozen storage and reheating. A total of 21 differential volatile compounds with variable importance in projection (VIP)≥1 were screened using the OPLS-DA model. Further ROAV analysis confirmed that compounds such as (E)-2-nonenal, dodecanal, and linalool were the key substances affecting the flavor quality of pre-cooked crayfish. It is concluded that the flavor characteristics of crayfish during storage could be enhanced by appropriately extending pre-cooking time. The composition of volatile flavor compounds was affected by both frozen storage duration and reheating time, prolonging both treatment times increased the variety of flavor substances. The results of this study can provide a scientific basis for flavor regulation of pre-cooked crayfish products during storage.