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High-speed imaging is very important in the semiconductor optical mask field and biology and life sciences. There have been countless auto-focusing methods based on reflected light for improving the imaging efficiency. However, these methods are not suitable for transparency or semi-transparency samples. There are some common characteristics for the samples, such as disorder and irregular for the structures on the sample surfaces transparency or semi-transparency. Focusing errors would occur due to the uneven reflectivity of the sample surface. This work presents a transmitted light spot method to solve the auto-focusing problem for the transparency or semi-transparency samples. The trace of transmitted light is simulated, and the expression of the focusing error signal is derived and given. The relationships between the defocus amount and focus error signal are calculated, and the results are experimentally demonstrated, accordingly. This method is expected to be applied to high-speed optical imaging.