Freeze-drying is often used as a final stage to produce three-dimensional porous matrices for medicine. Because a pure solvent crystallizes first during freezing, it acts as a pore-forming agent. The size of the solvent crystals primarily depends on the cooling rate and the composition of the material to be frozen. Ultrasonic treatment also affects the size of crystals and can be used to control the structure of a porous matrix. This article describes the effect of ultrasound (40 kHz, 50 W) applied at the preliminary freezing stage of polysaccharide solutions (alginate, chitosan, alginate–chitosan and alginate–gelatin) on the finished matrix properties. The most attention was paid to the effect of ultrasound on the size and shape of crystals formed during freezing, which leads to a change in the porous structure of the matrices after solvent sublimation. As a result of changes in the microstructure, a number of differences in the vibrational spectra of the molecules and the values of pore volume, sorption capacity, permeability and degradation of matrices were identified. Such changes in the structure of materials, as well as the emerging directionality of pores, together can affect the process of cell cultivation in these polysaccharide matrices, which can be useful in solving problems of tissue engineering.