A method of measuring area of leaves by reference object based on computer vision technology was studied. The sampling box of live leaves was developed, and its data were optimized. The measurement process was studied. The reference object was segmented from the original image by the method of threshold. The data of threshold was gained by means of derivation. The way of removing the noises in the image was studied. Generally, the results show that this method has better feasibility, higher precision and higher efficiency.