Abstract: Bio-printing can be described as an innovative technology that fabricates 3D functional multi-cellular tissue in vitro, namely deposited biological cells on biological scaffolds. Such efforts are being undertaken in many laboratories around the world. Agarose gel and its composite hydrogel are ideal material for scaffolds with superior biological, mechanical and structural properties. In order to research the optical properties of Agarose gel,which is applied to bio-printing, different concentration of Agarose gel were prepared by mixing up Agarose powder with distilled water in certain ratio after heating, dissolving and cooling. During the cooling procress, the refractive index of Agarose solution at 55℃ and Agarose gel was measured with Abbe refractometer. By comparing the refractive index of cell culture medium, the concentration of 0.00786 g/mL was proved to be the best concentration for Agarose hydrogel as bio-printing substrate materials and biological scaffolds. Visible transmittance spectrum, near infrared transmittance spectrum and attenuated total reflectance-Fourier transform infrared spectrum of Agarose gel and cell culture medium were measured, which shows that there is no absorption peak in the spectrum of agarose gel at the wave band from 400 nm to 1100 nm, and the maximum absorption is only 0.1711Abs; In the range of infrared spectrum, Agarose gel has the same absorption properties with cell culture medium, except for an unique absorption property at 2966 cm-1 in the spectrum of cell culture medium. The experiments of the laser scattering characteristic of the surface of Agarose gel show that the 440 nm laser has the strongest laser scattering intensity, while the 532 nm laser has the weakest one. Meanwhile, the smaller the concentration of Agarose gel, the stronger the scattering is.