Focus drift has a significant effect during long time live cell observations in microscopy imaging,so autofocusing is one of the most effective techniques to solve this problem.Active focus drift measurement using surface reflected signal is the key of auto-focusing,however,there is still a question to precisely measure the drift when multi-surface reflection exists.A measurement method of the focus drift for multi-surface reflection was proposed.The reflected spots were imaged by a linear array CCD,and the spots reflected from different surfaces can be easily separated on the linear array CCD.The weighted centroid method is used to accurately measure the drifts of the spots in defined window.The measurement precisions of the method are 106 nm and 111 nm under the condition of sample drying and wetting,respectively.