The present paper is a preliminary exploration of the possible way the gallstones are formed. Five categories of gallstones from clinical surgery in Xuzhou region were extracted by a series of solvents. Fourier transform infrared spectroscopy (FTIR) was used to characterize the structure of morphological changes between gallstone and residue by extracting. The gallstone samples are from clinical surgeries in Xuzhou region where gallstone disease is quite common. Samples were extracted with a series of solvents, and then FTIR and other instrumental analysis were applied to characterize the composition, structure and morphological changes of the samples both before and after the extraction. The results show that the gallstone samples can be classified as 5 types: cholesterol-type, cholesterol-based hybrid type with salt, bilirubin and protein as its insoluble substances, brown pigment type and black pigment type gallstones. The results also indicate that protein plays a key role in gallstone nucleation process by providing a sediment matrix for the formation of gallstones. Both cholesterol and carbonated hydroxyapatite are found in the insoluble substances of the samples. It is possible that cholesterol was accompanied by carbonated hydroxyapatite and there are interactions between them, and these interactions contribute to the crystallization process and speed up the formation of gallstones. All the results above may provide useful references for the clinical diagnosis, treatment and prevention of gallstones.