Secondary structure, intrinsic fluorescence emission spectra, micelle morphology and particle size distribution of bovine casein were researched respectively by circular dichroism, fluorescence spectrophotometer, scanning electron microscope and nano-particle size analyzer. Results showed that bovine casein by ultrahigh temperature sterilization (UHT) had more random structures(α-helix 22.1%, β-sheet 23.1%, β-turn 22.4%, random 33.4%), compared with casein by pasteurization(α-helix 47.4%, β-sheet 19.7%, β-turn 13.4%, random 19.4%). Tryptophan of casein by UHT had more hydrophobic. Casein particles by pasteurization were spherical with smooth surface, and able to connect to short micelles. But casein particle by UHT did not have smooth surface, and could not connect to micelles. Compared with pasteurization, casein particles by UHT was smaller, with the diameter being 221.7 nm. This research suggested the structure of casein by pasteurization and ultrahigh temperature sterilization had obvious differences.