Anchovy (Engraulis japonicus) is one of the most important species in the South Sea of South Korea. In order to estimate the biomass of anchovy, acoustic surveys, concurrently with trawling, were conducted in April, July, and September of 2013 and February of 2014 off the coast of Tongyeong and Yeosu. However, anchovies were found only during spring (April) and winter (February) surveys. To display only anchovy species confirmed by trawls on empty spaced echograms, virtual echograms were created. Three target strength (TS) values of anchovy were used: TS_z = 20 log L – (20/3) log (1+z/10) – 67.6 (Zhao et al.), TS_y =20 log L – 72.9 (Yoon et al.), TS_f = 20 log L – 71.9 (Foote). For anchovy schools combined with other species e.g., hairtail, squid, and Korean pomfret, their TS values were used to calculate an average TS. As a result, the anchovy biomass in the winter survey was approximately 75,000 tons (TS_z), 90,000 tons (TS_f), 114,000 tons (TS_y), respectively. The biomass in spring was 9,000 tons (TS_z), 9,200 tons (TS_f), 10,000 tons (TS_y). The sampling variance of spring was 70% and of winter was 30%. In order to manage anchovy resource sustainably, larvae and adult anchovy should be separately surveyed, the optimal area, date, and time for acoustic survey should be investigated, and the biomass survey should be done for relatively long and regular base.