Objective  To evaluate a custom-built dual channel dual focus spectral domain optical coherence tomography （OCT） instrument for imaging the whole eye in vivo， to demonstrate the feasibility and repeatability of this OCT instrument to measure anterior chamber depth and axial length compared to the IOLMaster. Methods  In a cross-sectional self-control study， 21 volunteers （39 eyes） without a history of ocular disease were enrolled. The anterior chamber depth （ACD） and total axial length （AL） were measured by the custom-built dual channel dual focus OCT and IOLMaster on the first day， and then by the OCT on the second day. The measurements of the two instruments were compared and the measurements with the OCT were compared for the 2 days. A paired t test， Pearson correlation analysis and Bland-Altman analysis were used for data analysis. Results  The ocular surfaces from the cornea to the retina could be assessed with this newly built dual channel dual focus spectral domain OCT in real time. The mean and standard deviation of the real anterior chamber depth and total axial length measured by the OCT system were 3.09±0.20 mm and 25.34±0.64 mm， respectively. No significant differences were found in the measurements with OCT taken on different days（t=-1.648， 1.129； P>0.05）. The mean and standard deviation of the anterior chamber depth and total axial length measured by IOLMaster were 3.58±0.21 mm and 25.24±0.65 mm， respectively. The mean and standard deviation of the anterior chamber depth by OCT was 3.64±0.20 mm. There was a significant difference in measuring the anterior chamber depth and total axial length with the OCT and IOLMaster （t=12.942， 8.984， P<0.05）. There was a high correlation in anterior chamber depth （r=0.990， P<0.01） and total axial length （r=0.997， P<0.01） measurements by the 2 devices. The 95% LoA （limits of agreement） were +0.001 mm to +0.119 mm and -0.018 mm to +0.178 mm for measurements of ACD and AL by the OCT and IOLMaster. Conclusion  Real-time whole eye images were successfully assessed by the custom-made dual channel dual focus OCT. There is good feasibility and repeatability with this newly built system.