Species identification in hydroacoustic survey is one of the key requirements to estimate biomass of organism and to understand the structure of zooplankton community. Feasibility of species identification using two frequencies (38 and 120 kHz) was investigated on the basis of mean volume backscattering strength difference (MVBS). Virtual echogram technique was applied to two frequencies data sets that obtained from surveys in the Antarctic Ocean and Yellow Sea. Virtual echogram method using MVBS revealed the possibility of species identification, which species identification relying on visual scrutiny of single frequency acoustic data resulted in significant errors in biomass estimation. Through noise cancellation using MVBS, much of the acoustic noise caused by acoustic instruments could be removed in new virtual echogram, and the biomass estimation and data quality was improved.