Formalin solution has been widely used to solidify the organization of articular cartilage and prevent tissue decomposition in long-time measurement. However, it was rarely investigated that the structural anisotropy changes of collagen fiber (fixation) of articular cartilage when it was immersed in formalin. In this paper, Fourier transform infrared spectroscopic imaging with polarization technique was used to investigate the anisotropic structure change of collagen fiber of articular cartilage fixed in formalin through the absorbance change of Amide I and Amide II with immersing time and polarization direction. The degree of anisotropy of collagen fiber in cartilage was characterized with fitting related coefficient of absorbance. The anisotropy of Amide I and Amide II became stronger with immersing extension of articular cartilage in formalin, and the amide I showed more remarkable anisotropy. It was concluded that the formalin solution induced new crosslinks of collagen, which gradually strengthened the collagen fiber anisotropy and was helpful for the structural analysis of the articular cartilage. The study will be significant for the preparation, preservation and anisotropy research of cartilage specimen.