ABSTRACT: Acoustic Doppler current profilers (ADCPs) have recently been used to estimate the dynamic characteristics and biomass of sound scattering layers (SSLs) or swimming speed of fish schools and to analyze SSL spatial distribution or various behavior patterns. This paper shows that it is necessary to verify mean volume backscattering strength (MVBS, dB) values acquired from each beam for quantitative analysis of the spatial distribution or the biomass estimates of such specific targets as SSL or a fish school when using an ADCP. In this study, the SSL was selected to be a homogeneous density layer over a large area and two methods were used to verify the MVBS values from each beam of the ADCP. First, a mutual comparison among four beams was conducted after calculating MVBS from the measured echo intensity. Second, the MVBS values were verified using comparison between the calculated MVBS from the 153.6 kHz ADCP and MVBS from three frequencies of a well-calibrated scientific echosounder. Moreover, the dominant scatterers (euphausiids) were collected by a framed midwater trawl. From these samples, biological data were used to identify the different frequency characteristics between two systems, using a distorted-wave Born approximation (DWBA) theoretical backscattering model in order to assess the averaged target strength and target strength TS differences for the three frequencies.