Mixed-mode chromatography has received more and more attention due to its unique chromatographic characteristics recently. In this review, a summary of the development and applications of mixed-mode chromatography is presented. According to the types of hydrophilic interaction/ion-exchange mixed-mode chromatography (HILIC/IEX), reversed-phase/hydrophilic interaction mixed-mode chromatography (RPLC/HILIC) and reversed-phase/ion-exchange mixed-mode chromatography (RPLC/IEX), the preparation and applications of each type are introduced. Compared with single mode chromatography, the selectivity as well as the sample loading capacity of the mixed-mode chromatography can be improved. The researches mostly focus on the design of mixed-mode stationary phases and the applications especially to bio-analysis. The mixed-mode chromatography will be a potent approach for the analysis and separation of complex samples, and it will also be an effective and complementary tool in practical separation work.