So far, systematic analysis of protein translation process is still far behind that of transcriptome. The ribosome profiling, based on deep sequencing of the ribosome-protected mRNA fragments, makes it possible to deeply and precisely study genome-wide protein translation process and translation regulation. This review introduces the development, construction and analysis of the ribosome profiling, and focuses on its wide applications. These applications include discovering novel ORFs (such as uORF and sORF), deciphering the mechanism of microRNA functional roles, charactering the translation efficiency and differential translation, predicting the protein abundance and illustrating the mechanism of protein translation. Finally, we analyze the broad development prospect of ribosome profiling.