Much of the current knowledge of human cardiovascular pathologies and treatment strategies has been gained from understanding the cardiac physiologies and functions in small animal models, such as mice, rats, and zebrafish. In this paper, we present the development of a high-frame-rate duplex ultrasound biomicroscopy (UBM) capable of B-mode imaging and pulsed-wave (PW) Doppler measurement for in vivo cardiovascular investigation in small animals. A frame rate of 200 frames per second (fps) was accomplished at a view of 5 mm x 8 mm, using a novel high-speed sector probe and specially designed lightweight transducers. In a reduced lateral view of 1.2 mm, a frame rate of 400 fps was achieved to examine more detailed cardiac motion. The UBM utilized transducers with different center frequencies (40-75 MHz) and geometries, which made it useful for various applications in small animal cardiac imaging. The highest spatial resolution the UBM achieved was 25 microm x 56 microm. In addition, the image-guided PW Doppler implemented in the UBM demonstrated the detection of the velocity of a moving wire as low as 0.1 mm/s, and flow in a polyimide tube as small as 200 microm in diameter. Furthermore, the UBM achieved a 15- microV minimal detectable signal and a 60-dB dynamic range using a low-cost PCB-based design. Finally, sample in vivo cardiac images of mouse and zebrafish hearts were given. These results showed that the UBM integrated with B-mode and PW Doppler is useful to investigate the pathophysiological mechanism in the cardiovascular studies.