Purpose-For some special manipulators such as the ones work at the space station, nuclear or some other unmanned environments, the overload, collision, vibration, temperature change or release of the internal stress would affect the structural parameters. And thus the operation precision might constantly decrease in long-term use. In these unmanned environments, the unattended manipulators should calibrate itself when they execute high precision operations or proceed self-maintenances. The purpose of this paper is to propose an automatic visual assistant on-line calibration (AVOC) method based on multi-markers. Design/methodology/approach-A camera fixed on the end of the manipulator is used to measure one to three identification points, which forms an unstable multi-sensor eye-in-hand system. A Gaussian motion method which combines the linear quadratic regulator control and extended Kalman filter together is proposed to make the manipulator track the planned trajectories when its inaccurate structural parameters form uncertain motion errors. And a Monte-Carlo method is proposed to form a high precision and stable signal acquisition when the visual system has measurement errors and intermittent signal feedback. An automatic sampling process is adopted to select the optimal measurement points basing on their variances. Findings-Data analysis and experiment results prove the efficiency and feasibility of the method proposed in this paper. With this method, the positioning accuracy is largely promoted from about 2 mm to 0.04-0.05 mm. Originality/value-Experiments were carried out successfully on a manipulator in a life sciences glove box that will work at the Chinese space station. It is a low cost and efficient manipulator calibration method. The whole autonomic calibration process takes less than 10 min and requires no human intervention. In addition, this method not only can be used in the calibration of other unmanned articulated manipulator that works in deep ocean, nuclear industry or space but also be useful for the maintenance work in modern factories owing a lot of industrial robots.