Ultrasonic backscatter signal is quite sensitive to the microstructure of cancellous bone. Trabecular bone spacing (TbSp) is an important parameter for characterizing bone microstructures. In order to acquire TbSp accurately from the ultrasonic backscatter signal of the cancellous bone, a TbSp estimation method is proposed using a combination of the Hilbert transform and fundamental frequency estimation (HFE) method. The HFE results from the cancellous bone in vitro are compared with the TbSp obtained from μ-CT. The HFE results are accurate (estimation error<3%) and stable (standard variation<4%) at higher frequencies (5 MHz and 10 MHz), and more accurate when standard TbSp is large, and have a high correlation (r2=0.75—0.99, p<0.01, n=16) with the standard TbSp at different frequencies. It shows that the HFE method is accurate and stable for TbSp estimation. The HFE method is vertified to characterize the cancellous bone TbSp.