Abstract: According to the acoustic survey data of squid (Sthenoteuthis oualaniensis) in the South China Sea in 2013 obtained by Simrad EK60 (38 kHz, 120 kHz) and Simrad EY60 (70 kHz, 120 kHz) scientific echosounders, we investigated the target strength (TS) and spatial distribution of squid, interference of zooplankton, potential of dB difference technique in the acoustic identification. The results reveal that the TS of squid could be measured by in situ method with light-falling net as biological sampling device. Squid echo was not obvious under natural light condition, but the density of squid increased significantly when the light was on. Before 22: 00, the squids mainly assembled in layers at 10~50 m and 55~80 m, while after 22: 00, the squids assembled in layers at 10~35 m, 50~75 m and 115~155 m. Zooplankton and pelagic fish caused considerable interference in the acoustic estimation especially at night, so different thresholds should be selected during different time periods. The dB difference technique which might be important in discriminating squid echogram should be studied further.