Tilapia production is one of the most diversified fish sectors globally and is expected to continually grow. In Taiwan, Nile tilapia (Oreochromis niloticus) is the second most produced fish species in the aquaculture industry. Traditionally, fishermen would use a net to estimate the volume of the fish species in the, pond but this method offer numerous disadvantages and acoustic methods were proven to be reliable in fisheries resource survey. Thus, this study explored an acoustic method to measure the standing stock and biomass in a Nile tilapia aquaculture pond. A rectangular tank (2.3 × 0.82 × 0.66 m) with a set of echo sounders (frequencies: 120 and 200 kHz) fixed on a steel frame was utilized to approximate the target strengths of Nile tilapia samples (n = 35) in lateral positions. Findings reveal that the estimated target strength using the two beam frequencies did not differ significantly (p > 0.05). A horizontal conversion equation in lateral position was established, which was used for biomass backscattering analysis of Nile tilapia in fish ponds. The results show that 120 kHz frequency can accurately predict Nile tilapia fish biomass in an aquaculture pond. This study has determined a species‐specific equation for the hydroacoustic method to enable a more accurate estimation of fish biomass, such as that of Nile tilapia. Fish biomass estimation will enhance aquaculture management, such as optimizing capital invested and minimizing overfeeding.