Abstract Modern challenges in the increasing exploitation of aquatic ecosystems require efficient, reliable, and noninvasive technologies to acquire biomass information on a large scale. For the past 40 years, hydroacoustics has been an essential tool to analyse fish populations and their relationship with the environment. Currently, split-beam echo sounders are standard tools used to reliably and accurately record data in oceans, estuaries, and lakes. To maximize the coverage volume and to increase target detection, and therefore data quality, the use of multi-beam echo sounders is a real asset. We propose here an innovative method for target strength (TS) calculation based on the signal from a reversible Mills cross multi-beam sonar, SeapiX (Exail), which also includes the analytical capability of a split-beam echo sounder. This innovative approach provides new original information when using a multi-beam sonar. The case study in Lake Bourget was based on a comparison of the simultaneous recordings of SeapiX and EK80 (SIMRAD) to prove the validity of this multi split-beam processing, as well as to estimate the in situ TS of fish.