In order to test the variability in color discrimination among observers with normal color vision and study the spectral responses of cone cells in R, G, B three channels, the spectral pseudoisochromatic images consisting of background layer, lightness layer and number layer, which have different spectral reflectance were designed and created. By interacting with the light source and the spectral response of the human cone cells, different observers perceive various color differences. The obtained visual results were used to further study the difference of human perception on the metameric color pairs. The experiments were based on different output equipments with different primary inks producing different spectral reflectances, and the metameric color pairs were created under different light sources, which can amplify the observer’s variability. For the CIEDE2000 color difference calculated by different color matching functions, some were within the threshold of color discrimination, and some could be clearly recognized. By optimizing calculation, the nearly metameric color pairs, which can magnify the color discrimination of observers, were applied to design the number and background layers of the spectral pseudochromatic image. The Epson inkjet printer and OKI laser printer were used to print the number layer and background layer of the pseudochromatic spectral image correspondingly. 72 color-normal observers, including 55 young observers aged from 18 to 25 and 17 old observers aged from 62 to 74, were organized to view and test the images under D65 and LED-5000K light sources respectively. The visual results showed that the spectral pseudochromatic images can be successfully used to classify whether the spectral response of retinal cone cells is aging or not. Under the same light source, young observers can read the numbers in images (②/④) that the old observers can’t read, while the old observers can read the numbers in images (①/③) that most young observers can’t read, and 4 of the 55 young observers have the same visual results as the elderly. In addition, the spectral responses of young observers were consistent with the CIE1964 and CIE2006 (age=25 y) Color Matching Functions(CMFs), while the spectral response of old observers were more consistent with the CIE1931 and CIE2006 (age=75 y) CMFs. It was found that the cone cell spectral responses of old observers were shifted to the long wavelength, and the spectral responses decreased due to the increase of the optical density of the ocular system.