Japanese anchovy (Engraulis japonicus) is one of important species, as the fisheries resources and ecological niche, in the coastal areas of the northwestern Pacific Ocean. Hydroacoustic ex situ target strength (TS) measurements of the live fish were made using 38, 120, and 200 kHz split-beam echo-sounder. The study examined 35 anchovy from small and adult size; total length ranged from 4.8 to 12.2 cm. TS experiments has been conducted under the tethered method with very small hooks that were attached to the mouth. Simultaneously with the acoustic measurements, underwater video camera was used to continuously monitor behavior and tilt angle. The standard fit regressions of TS on total length (TL, cm) were follows: TS38kHz= 20 log [TL] – 65.8 (r=0.91), TS120kHz = 20 log [TL] – 68.4 (r=0.92), and TS200kHz = 20 log [TL] – 69.1 (r=0.84). In case of fish wet weight W (g), best fit equations were follows; TS38kHz = 5.1 log [W] – 50.5 (r=0.92), TS120kHz = 4.9 log [W] – 53.0 (r=0.95), TS200kHz = 4.6 log [W] – 53.6 (r=0.89). Additionally, the mean swimming angle and standard deviation were 9.1 13.1. The results of ex situ measurements provided essential information for studying TS of Japanese anchovy for acoustic survey and thus improve the accuracy of anchovy biomass estimates.