Objective To investigate platelet aggregation on glass surface under physiological flow condition for establishing a simple and novel new model for platelet function analysis under flow condition. Methods The polydimethylsiloxane (PDMS) -glass microchannel chips were fabricated by soft lithography based on a photosensitive dry film. Anti-coagulant human peripheral whole blood was flowed through the microchannel chip at a flow shear rate of 300 s^-1 and 1500s^-1. The fluorescence images of platelet aggregates formed on the 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 value (Hct) of the blood sample was adjusted, and the whole blood was treated with different anti-platelet agents. The platelet aggregation on the glass surface was observed under the above experimental conditions. The platelet aggregations in healthy people and diabetic patients were also analyzed. Results The results showed that the platelet aggregation on the glass surface under the flow condition was three-dimensional. Platelet aggregation was dependent on shear rate, glass surface hydrophilicity 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. Conclusion Platelet aggregation on glass surface under flow condition is related to flow condition, protein adsorption and platelet activation state. This method might provide a new model for the detection platelet aggregation by microfluidics chip in vitro without additional adhesion protein modification.