A simple, selective and rapid method for determination of ascorbic acid from fruit juices was developed by combining a flow injection analysis (FIA) system with a chemiluminometric detector and a reactor with L-ascorbate oxidase immobilized on controlled pore glass. It was found that some reducing agents (eg ascorbic acid and mercaptoacetic acid) give chemiluminescence with luminol in the presence of hexacyanoferrate (III) in an alkaline solution. We used this new type of chemiluminescent reaction for the enzymatic determination of ascorbic acid. The background substraction method was used in order to avoid interference during ascorbic acid determination. Accordingly, two chemiluminometric signals were registered for each determination, one signal corresponding to the sample that passed through the enzymatic reactor that decomposed the ascorbic acid completely, and the second signal corresponding to the sample that does not pass through the reactor. The difference between the two signals corresponds to ascorbic acid from the sample. The linear range of the method was 10-1000 micromol/L of ascorbic acid and the detection limit was 5 micromol/L The throughput was four samples/h and RSD 3.13% (n = 10). This method was applied for determination of ascorbic in fruit juices. The results were compared with those found by the reference method, based on titrimetric determination with 2,6-dichlorophenolindophenol, and the concordance was excellent.