This study aimed to determine and explain changes in the reflectance spectrum of human blood in vitro when exposed to laser radiation at wavelengths of 450 or 980 nm. Reflectance spectra of venous blood samples were measured before and after exposure to a single pulse of 450 or 980 nm laser radiation. A numerical optical model based on the Monte Carlo method was applied. Laser irradiation at 450 and 980 nm caused the most significant changes in the reflectance spectrum around 600 nm, associated with alterations in blood oxygen saturation. The maximum efficiency of reducing oxygen saturation was 0.20%/W for 980 nm and 0.72%/W for 450 nm, likely due to differences in blood absorption at these wavelengths. The greatest change in intensity reflectance spectra and oxygen saturation of human venous blood occurs when exposed to laser radiation at 450 nm, not at 980 nm.