Microflow cytometers and many other miniaturized microfluidic devices have shown great potential in many fields, such as, particle detection, cell sorting and classification. A reliable signal analysis method is required to improve the measurement accuracy of the emerging microfluidic devices. In this paper, a novel method is presented to analyze the signal from microspheres with different diameters based on transit time and amplitude. Experimental results show that transit time threshold plays a more important role at lower flow rate for particle differentiation and can be used to improve the performance of a microflow cytometer.