A high-accuracy Winkler titration method has been developed for determination of dissolved oxygen concentration. Careful analysis of uncertainty sources relevant to the Winkler method was carried out and the method was optimized for minimizing all uncertainty sources as far as practical. The most important improvements were: gravimetric measurement of all solutions, pre-titration to minimize the effect of iodine volatilization, accurate amperometric end point detection and careful accounting for dissolved oxygen in the reagents. As a result, the developed method is possibly the most accurate method of determination of dissolved oxygen available. Depending on measurement conditions and on the dissolved oxygen concentration the combined standard uncertainties of the method are in the range of 0.012–0.018 mg dm−3 corresponding to the k = 2 expanded uncertainty in the range of 0.023–0.035 mg dm−3 (0.27–0.38%, relative). This development enables more accurate calibration of electrochemical and optical dissolved oxygen sensors for routine analysis than has been possible before.