Zooplankton can be detected by using acoustic Doppler current profiler (ADCP) instruments through acquiring the mean volume backscattering strength (MVBS) data. However, the precision of the backscattered signal measured by single ADCP measurement has a limitation in the MVBS variation of zooplankton. The objectives of this study were to analyze the MVBS and vertical velocity from ADCPs at the same time and location for zooplankton’s daily vertical migration (DVM) observation. Measurements were conducted in Lembeh Strait, North Sulawesi, Indonesia. Instruments used included a moored ADCP 750 kHz and a mobile ADCP 307.2 kHz. High MVBS value was found at 11.5–16 m depths and was identified as the sound scattering layer (SSL). The DVM patterns in the SSL displayed significant differences over time and had good relationships with the diurnal cycle. Theoretical target strength (TS) from the scattering models based on a distorted-wave Born approximation (DWBA) was estimated for Oithona sp. and Paracalanus sp.; the two dominant species found in the observed area. However, ΔMVBS and ΔTS proved that the dominant zooplankton species were not the main scatterers. The strong signal in SSL was instead caused by the schools of various zooplankton species.