A new method using a double-sensor difference based algorithm for analyzing human segment rotational angles in two directions for segmental orientation analysis in the three-dimensional (3D) space was presented. A wearable sensor system based only on triaxial accelerometers was developed to obtain the pitch and yaw angles of thigh segment with an accelerometer approximating translational acceleration of the hip joint and two accelerometers measuring the actual accelerations on the thigh. To evaluate the method, the system was first tested on a 2 degrees of freedom mechanical arm assembled out of rigid segments and encoders. Then, to estimate the human segmental orientation, the wearable sensor system was tested on the thighs of eight volunteer subjects, who walked in a straight forward line in the work space of an optical motion analysis system at three self-selected speeds: slow, normal and fast. In the experiment, the subject was assumed to walk in a straight forward way with very little trunk sway, skin artifacts and no significant internal/external rotation of the leg. The root mean square (RMS) errors of the thigh segment orientation measurement were between 2.4 degrees and 4.9 degrees during normal gait that had a 45 degrees flexion/extension range of motion. Measurement error was observed to increase with increasing walking speed probably because of the result of increased trunk sway, axial rotation and skin artifacts. The results show that, without integration and switching between different sensors, using only one kind of sensor, the wearable sensor system is suitable for ambulatory analysis of normal gait orientation of thigh and shank in two directions of the segment-fixed local coordinate system in 3D space. It can then be applied to assess spatio-temporal gait parameters and monitoring the gait function of patients in clinical settings.