The aim of this study was to establish three dimension high frequency ultrasound computed tomography (HFUCT) for detecting the testicular tumor in vitro. Currently, the UCT has been applied for diagnosing the breast cancer for females. This study is the first report for applying this modality for testicular tumor applications. Experiments were carried out by artificial testicular tumor from rat. Two 25 MHz transducers were used in this research. The transmitting transducer and receiving transducer were both fitted on the three-axes step motor platform by two brackets, and the testicle was fixed on a rotational stage which was controlled by a rotating motor. The projection data of UCT were obtained by measuring the ultrasonic attenuation. A filtered backprojection algorithm was used to reconstruct the HFUCT images. In the results of healthy testicle, the transmission attenuation HFUCT image and the B-mode imaging both showed that the testicular tissue had a homogeneous property because the distribution of ultrasonic attenuation seems uniform within whole testis. In the results of testicular tumor samples, it was difficult to detect the tumor by using B-mode image, however, the size and location of tumor was observed clearly by the HFUCT image. Moreover, the three dimensional HFUCT image was reconstructed in this study, and the volume of tumor can be measured accordingly.