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Nowadays, silicon nanoparticles (Si-NPs) are extremely promising for use in biophotonics, due to a combination of specific structural and optical properties with high biocompatibility, bioavailability, biodegradability and low toxicity of the material. In this work, we study the possibility of the formation of Si-NPs by picosecond laser ablation of silicon nanowires (Si-NWs) in distilled water and ethanol. In this case, the Si-NWs were previously formed by the method of metal-stimulated chemical etching of single-crystal low- and high-dopped silicon wafers. As a result of applying the two-stage technique, it becomes possible to obtain a suspension of chemically pure Si-NPs, promising for use in biomedical applications. Thereafter a research was made of the structural and optical properties of the formed samples. Using atomic force microscopy, it was found that the ensembles of the obtained Si-NPs have a size in the range of 10–90 nm. It should be noted that the peak in the histogram of particle size distribution is in the range of 30–40 nm. The measurements made by Raman scattering indicate the formation of crystalline silicon. Thresholds of ablation of Si-NPs are determined. For all cases, the ablation threshold is significantly lower than this value for single- crystal silicon, which allows one to obtain a larger number of Si-NPs at lower energy costs. Photoluminescence (PL) studies show that the formed structures exhibit effective PL with an emission peak falling into the diagnostic window of transparency of biological tissues. This fact and the relatively small size suggest that Si-NPs can be used as PL-markers in living organisms for the diagnosis of various diseases.