Flow injection spectrofluorimetry with in-line Winklers procedure was developed for the dissolved oxygen (DO) determination. 2-Thionaphthol reacted with iodine produced by Winkler's method to form fluorescence inactive disulfide compound. To automate the process completely, a 5-channel flow system with a newly designed 16-way valve was assembled. The system consisted of a dispersion coil (DC), a precipitate formation coil (PFC), a precipitate dissolving coil (PDC), and extraction coil (EC). A calibration can be constructed by using a standard iodine solution for dissolved oxygen. The calibration graph was linear over the range 1.2×10-4∼6.0×10-4 mol l-1 iodine (1.96∼9.80 mg O l-1)). The relative standard deviation (n=6) was below 0.3% for the 4×10-4 mol l-1 iodine (6.27 mg O l-1) determination. The sample throughput was 12/h. © 2004 Elsevier B.V. All rights reserved.