A continuous system for the determination of fish freshness with double enzyme reactors was developed and applied to the determination of the freshness indicator (Ki = [(HxR + Hx)/(IMP + HxR + Hx)] X 100) in many types of fish, where IMP, HxR and Hx are inosine monophosphate, inosine and hypoxanthine, respectively. The system was prepared from two combinations of oxygen electrodes and reactors. One reactor for the determination of the total amount of HxR and Hx was packed with nucleoside phosphorylase (NP) and xanthine oxidase (XOD) immobilized simultaneously on chitosan porous beads. Similarly, another reactor for IMP, HxR and Hx was packed with 5nucleotidase (NT), NP and XOD immobilized simultaneously on chitosan beads. The system was prepared from two combinations of oxygen electrodes and reactors. One assay could be completed within 5 min. The system for the determination of fish freshness was reproducible within 2.1% (n = 30). The immobilized enzymes were sufficiently stable for at least 7 months at 4ยฐC. More than 200 samples could be analysed in about 1 month by using these enzyme reactors. The results for fish meat (13 types) correlated satisfactorily with those obtained by liquid chromatography (r = 0.989, n = 253) and ion-exchange column chromatography (r = 0.973, n = 50). These results suggest that the proposed sensor system provides a simple, rapid and economical method for the determination of fish freshness (Ki).