In order to test the capture ability of holographic optical tweezers, an optical measurement system based on vision theory was used to measure the 3D trap stiffness of holographic optical tweezers. In the optical system, two symmetrically distributed light sources irradiated the sample cell to form two images. The 3D positions of the microspheres captured by the holographic optical tweezers were obtained by tracking the displacements of their images. Combined with hydrodynamic method, the trap stiffness of the single and double optical traps was measured. The experimental results show that in a single optical trap, there is little difference between X and Y horizontal trap stiffness, and the axial trap stiffness falls in the range of 1/3 to 1/4 of the horizontal direction; in double optical traps, the three-dimensional stiffness of the optical trap near the optical axis is larger than that an a far distance, which demonstrates that the closer to the diffraction center, the higher the diffraction efficiency of light, and the stronger the capture ability of the optical trap.