With the development of science and technology, more and more complex samples like peptides and proteins need to be separated. It is difficult to separate them by single mode chromatography. Due to the unique separation character of mixed-mode chromatography, the same separation performance can be obtained in mixed-mode chromatography in one separation as multidimensional chromatography. And some disadvantages of multidimensional chromatography can be avoided in mixed-mode chromatography, such as the complexity of the system, the poor compatibility of mobile phases and the long analytical time. More and more attention is devoted to the mixed-mode chromatography in recent years. The focus on the mixed-mode chromatography is to design new structures of mixed-mode stationary phase. At present, mixed-mode stationary phases included reversed-phase/ion-exchange, reversed-phase/hydrophilic, hydrophilic/ion-exchange, zwitterionic and trimode mixed-mode stationary phases. The kinds of mixed-mode stationary phases are summarized and their features and applications in different fields are discussed in this review.