The intensification of conflicts associated with the use of water in the transition region of the Cerrado and Amazon biomes caused by population and economic growth, combined with the interest in generating energy from hydroelectric plants, raise the need to quantify the surface water availability of rivers contributing with different drainage areas. The present study estimated and compared in loco measurements of liquid flow (QL) and the depth of rivers in the Teles Pires river basin by reference methods (MLN-7 hydrometric windlass and metal rod/winch) and by Acoustic Current Profiler by Doppler Effect (ADCP RiverRay), in this last method the uncertainty estimate of the total measurement time by ADCP was evaluated. Field measurements were carried out at monthly intervals between March 2020 and October 2021, seeking to represent the water seasonality and depth and QL variations in the cross-sections of the Caiabi 1 and 2, Celeste, Preto and Renato rivers. The evaluated rivers had a net flow between 3.48 and 60.78 m3 s−1 by the windlass and between 2.66 and 54.30 m3 s−1 by the ADCP, while the depths obtained were from 0.17 to 6.34 m by the rod/winch and from 0.65 to 6.20 m by the ADCP. The methods resulted in similar measurements of net flow and depth in each of the cross-sections, and the statistical performance of the linear regression model was satisfactory with a Willmott concordance index of 0.9977 and 0.9819 for estimates of QL and of the depth of the cross-sections, respectively. The ADCP accurately measured the net discharge and depth in shallow (up to 6.5 m) cross-sections of the Teles Pires River relative to the reference method. Determining the total measurement time and pairs of transects to obtain accurate QL by ADCP depends on the hydraulic characteristics of the watercourses.