The invention discloses a device and a method of assembly of a microtubule and a microsphere based on monocular microscopic vision. The device of the assembly of the microtubule and the microsphere based on the monocular microscopic vision comprises a motion platform, a manipulator, a clamping device, a translational motion platform, a microscopic vision system, a support frame and a vibration isolation platform, wherein the microscopic vision system is installed on the translational motion platform, the translational motion platform is installed on the support frame, the microtubule is installed on the manipulator through the clamping device, the microsphere is placed on the motion platform, and the support frame, the manipulator and the motion platform are installed on the vibration isolation platform. When the microtubule and the microsphere are assembled, the motion platform is adjusted so that the microsphere is enabled to enter a view of the microscopic vision system, the translational motion platform is adjusted so that an image of micropore formed in the microsphere is enabled to be clear, the microtubule is driven by the manipulator to enter the view of the microscopic vision system, the manipulator is adjusted till the tail end of the microtubule is clear, the manipulator drives the microtubule to conduct a three-dimensional motion, and the microtubule is driven to be align with and be inserted into the micropore. According to the alignment and assembly device and the method of the microtubule and the microsphere based on the monocular microscopic vision, the monocular microscopic system is utilized to achieve alignment and assembly of the microtubule and the microsphere without the need of repeated focusing of the microscopic vision system, and the application is convenient.