Fourier transform infrared (FTIR) microspectroscopy was applied to in-situ analyse the chemical change of tibial articular subchondral bone of female Hartley guinea pigs with age increase. Three infrared absorption regions (a,b,c) of trabecular bone and central marrow region of the subchondral bone were measured for guinea pigs of different ages (1 months, 2 months and 3 months) using the infrared spectrum. Results show that (1) with months increasing, the total area of trabecular bone is increasing, meanwhile, the region a which is similar to normal trabecular bone spectra is decreasing, and region d waveform has the same trend as region a.(2) In the second and third month, region b & c show amide Ⅲ redshift and the red shift in region c shows a shoulder peak, showing the absorption peak intensity on behalf of nucleic acid and polysaccharide in region b & c is 7 times that in region a.(3)β glycosidic bond absorption peak appears at region c in 3 different old pigs.(4) IamideⅢ/IamideⅡ is the highest in region b in the second month but lowest in the third month; IamideⅢ/IamideⅡ reduces from a to c in the second and third month; IνsPO-2/IamideⅡ in region b & c is 7 times higher than region a in the second and third Month. These results are consistent with the regular pattern of change rule of osteoarthritis subchondral bone’s organization structure and chemical composition in different stages . Our primary result illustrated that FTIR microspectroscopy can be used for in-situ analysis of the molecular organization of subchondral trabecular bone and bone marrow. It provides reliable pathology information for osteoarthritis subchondral bone tissue at molecular level.