Acetic acid fermentation of pineapple vinegar was processed by self-priming fermenter, and this study aimed to show the relationship between flavor and metabolic substances in industrial production. E-nose and E-tongue respectively characterized the changes in aroma and taste. Meanwhile, the organic acids, free amino acids, volatile components were monitored in real time by HPLC and HS-SPME-GC-MS. The results show that the most significant flavor changes took place in the initial stage (0 - 4 days). The floral aromas initially provided by ethanol diminished, was replaced by the emergence of rich fatty and fruity aromas attributed to acetic acid and Ethyl-2-methylbutanoate, respectively. The bitter of pineapple vinegar was reduced, acidity and astringency were significantly increased, which may be due to changes in amino acids and organic acids. In addition, the DPPH radical and FRAP scavenging activity remain stable, while HFRC radical scavenging activity increased significantly in pineapple vinegar, reached 55.78%. Our study findings provided valuable information on the functional characteristics and quality of pineapple vinegar.