This study measures the dorsal aspect target strength (TS; dB re 1 m2) and TS–length (standard length, SL; cm) relationships for European anchovy, attained both ex situ and in situ in two different seasons across 7 years in the Bay of Biscay. The measurements were made at three frequently used acoustic frequencies (38, 120, and 200 kHz). A backscattering model for physostome fish was utilized to help interpret the results. The obtained experimental mean TS for anchovies with an SL of 3.5–19.5 cm was −44.6 (±2.3), −46.9 (±3), and −48.4 (±2.7) dB at 38, 120, and 200 kHz, respectively, yielding b20 values of −66.4, −68.7, and −70.4 dB, respectively. The results were consistent across seasons and between in situ and ex situ conditions, presenting TS–length relationships with statistically significant slopes (p-values <0.05) for all frequencies. This research represents part of a series of efforts planned to obtain a comprehensive TS vs. length and depth relationship to update the acoustic assessment methodology of European anchovy in the Bay of Biscay.