Objective To investigate platelet aggregation on glass surface under physiological flow condition. Methods The polydimethylsiloxane (PDMS)-glass microchannel chips were fabricated by soft lithography. Anti-coagulant human peripheral whole blood was flowed through the microchannel chip at flow shear rate of 300 s-1 and 1 500 s-1, respectively. The fluorescence images of platelet aggregates formed on glass surface at the bottom of the microchannel were captured after 150 s using an inverted fluorescence microscope. The number of platelet aggregates, average size, surface coverage and average fluorescence intensity were quantified by image analysis. The glass surface was treated with oxygen plasma, BSA blocking or collagen modification to establish different surfaces for platelet aggregation. The hematocrit (Hct) of blood sample was adjusted, and the whole blood was treated with different anti-platelet agents. The platelet aggregation on glass surface was observed under the above experimental conditions. The platelet aggregations in healthy people and diabetic patients were also analyzed. Results Under the flow condition, platelet aggregation on glass surface was three-dimensional. Platelet aggregation was dependent on wall shear rate, the hydrophilicity of glass surface and Hct, and was mainly regulated by GPIIb/IIIa-fibrinogen and ADP-P2Y12 receptor pathways. The aggregation of platelets on the glass surface could also reflect the high activity of platelets in diabetic patients. Conclusions At the flow conditions of 300 s-1 and 1 500 s-1, platelet aggregation on glass surface is related to flow rate, protein adsorption, platelet related receptors and platelet activation state. In this study, a new model for microfluidic platelet function analysis without additional adhesion protein modification was established, and it could be used for clinical evaluation of platelet function.