Acoustic volume backscattering strength (SV) was measured at frequencies of 38, 50, 120 kHz from zooplankton and juvenile fish in the sound scattering layer (SSL) off northwestern Hokkaido. Organisms in the SSL were collected by beam trawl hauls at eight stations where the acoustic measurements were recorded. Euphausiids, copepods and juvenile walleye pollock were abundant organisms in the SSL. The composition of the species of sampled organisms was different at each station. When euphausiids were dominant in the SSL, the SV increased with acoustic frequencies. When juvenile walleye pollock were dominant, the SV showed a higher value than that in the above situation, and it was less changed by acoustic frequencies. These differences were explained by the frequency characteristics of acoustic backscattering of euphausiids and walleye pollock. The target strengths of euphausiids and juvenile walleye pollock were estimated by the multiple linear regression model. The results showed that there were some differences between estimated TS and theoretical TS, and the possibility of estimating TS using the multiple linear regression model.