To measure and characterize the roughness spectrum of sea-bottom, which is an important input parameter of the bottom acoustic backscattering model, an underwater laser three-dimensional (3D) scanning system for measuring seafloor microtopography has been developed successfully. The proposed underwater laser 3D scanning system can provide 3D seafloor microtopography with the resolution of millimeter levels in both horizontal and vertical directions and obtain the roughness spectrum of sea-bottom further. Two sets of data, which were suitable to model low- and mid-frequency acoustic backscattering at 6–24 kHz, were collected in two sites of sandy bottom during the acoustic scattering experiment in the southern Yellow Sea. The one-dimensional (1D) and two-dimensional (2D) roughness spectrum of the seafloor have been calculated at the two sites named S1 and S2, respectively. The result shows that calculated 2D spectral indices and spectral intensities, respectively, were 3.45 and 2.17 × 10−4 m4 at S1 and 3.33 and 4.66 × 10−4 m4 at S2. 2 D roughness spectrum parameters were used to model the acoustic scattering at S1, and the result indicates that the measurement results of the seafloor roughness spectrum is suitable for modeling bottom acoustic scattering.