We have developed a flow cytometer based on simultaneous detection of ultrasound and photoacoustic waves from individual particles/cells flowing in a microfluidic channel. Our polydimethylsiloxane (PDMS) based hydrodynamic 3-dimensional (3D) flow-focusing microfluidic device contains a cross-junction channel, a micro-needle (ID 100 μm and OD 200 μm) insert, and a 3D printed frame to hold and align a high frequency (center frequency 375 MHz) ultrasound transducer. The focused flow passes through a narrow focal zone with lateral and axial focal lengths of 6-8 μm and 15-20 μm, respectively. Both the lateral and axial alignments are achieved by screwing the transducer to the frame onto the PDMS device. Individual particles pass through an interrogation zone in the microfluidic channel with a collinearly aligned ultrasound transducer and a focused 532 nm wavelength laser beam. The particles are simultaneously insonified by high-frequency ultrasound and irradiated by a laser beam. The ultrasound backscatter and laser generated photoacoustic waves are detected for each passing particle. The backscattered ultrasound and photoacoustic signal are strongly dependent on the size, morphology, mechanical properties, and material properties of the flowing particles; these parameters can be extracted by analyzing unique features in the power spectrum of the signals. Frequencies less than 100 MHz do not have these unique spectral signatures. We show that we can reliably distinguish between different particles in a sample using the acoustic-based flow cytometer. This technique, when extended to biomedical applications, allows us to rapidly analyze the spectral signatures from individual single cells of a large cell population, with applications towards label-free detection and characterization of healthy and diseased cells.