This paper presents an image-based visual servoing algorithm to perform the microassembly process with an uncalibrated manipulator. The proposed algorithm requires only the use of the visual information from a single-vision camera to evaluate the unknown Jacobian matrix. Two methodologies were examined to estimate the Jacobian matrix on-line. Through monitoring the selected feature and the image reflection from the surface, the 3D position between the slot and the micropart can be evaluated successfully for the assembly process. Experimental results confirmed that the Jacobian matrix computed from both methods can evaluate the position with an accuracy of 3.6 µm initially. By using a proportional gain control, the position accuracy can be improved to within 1 µm. Measurement noise during the image acquisition is determined to be one of the root causes of the evaluation accuracy.